Compositions having antiangiogenic activity and uses thereof

ABSTRACT

The invention generally features compositions and methods that are useful for modulating blood vessel formation, as well as methods that provide for the systematic and efficient identification of angiogenesis modulators. As described in more detail below, a systematic computational methodology based on bioinformatics was used to identify novel peptide modulators of angiogenesis that have been characterized in vitro and/or in vivo.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.11/992,001, filed Apr. 2, 2009, which is a U.S. National Stageapplication of PCT/U06/35580, filed Sep. 12, 2006, which claims thebenefit of U.S. Provisional Application No. 60/716,341, filed Sep. 12,2005. Each of these are hereby incorporated by reference in itsentirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by the following grant from the NationalInstitutes of Health, Grant No.: HL79653. The government may havecertain rights in the invention.

BACKGROUND OF THE INVENTION

Angiogenesis, the process of developing a novel vascular network from apre-existing one, is tightly controlled by various endogenousregulators. These regulatory elements include both pro- andanti-angiogenic proteins that finely modulate the neovascularmorphological and functional characteristics. Where the regulation ofsuch processes is disrupted a variety of pathological conditions canensue, including neoplasia, hematologic malignancies, rheumatoidarthritis, diabetic retinopathy, age-related macular degeneration,atherosclerosis, endometriosis, pathologic obesity, and ischemic heartand limb disease. An urgent need exists for angiogenesis modulators thatcan be used as therapeutics for these and other numerous angiogenesisrelated diseases and conditions. While some promising angiogenesismodulators have been identified, to date, the quest for the experimentalidentification of such agents has been an empirical time-consumingprocess. Improved angiogenesis modulators and methods for systematicallyidentifying and assessing the biological activity of such agents areurgently required.

SUMMARY OF THE INVENTION

As described below, the present invention generally featuresangiogenesis modulators, related prophylactic and therapeutic methods,as well as screening methods for the identification of such agents.

In one aspect, the invention generally features an isolated peptide oranalog thereof containing or consisting essentially of the amino acidsequence W—X₂—C—X₃—C—X₂-G (SEQ ID NO: 161) that reduces blood vesselformation in a cell, tissue or organ. In various embodiments, thepeptide contains at least 11, 12, 15, 20, 25, 30, 35, or more aminoacids of a thrombospondin-1 domain or thrombospondin-2 domain.

In another aspect, the invention features an isolated peptide or analogthereof containing or consisting essentially of a 20 amino acid (AA)sequence having positions AA1-AA20 (SEQ ID NO: 162), wherein:

AA1 is X, S, T, G, Q, or A;

AA2 is X, P, E, S, A, Q, or K;

AA3 is W;

AA4 is X, S, T, G, E, D, R, or A;

AA5 is X, P, A, Q, D, E, K, R, or V;

AA6 is C;

AA7 is X, S, N, or T;

AA8 is X, V, A, R, K, G, S, T, or E;

AA9 is X, T, S, R, or N;

AA10 is C;

AA11 is X, G, S, or N;

AA12 is X, G, K, R, M, T, L, D, S, or P;

AA13 is G;

AA14 is X, V, I, M, T, H, A, E, F, K, R, S, Q, W, or Y;

AA15 is X, Q, S, R, K, Y, or A;

AA16 is X, T, F, K, Q, S, L, E, M, N, or V;

AA17 is X, R, S, or Q;

AA18 is X, S, T, V, R, H, E, Q, A, or I;

AA19 is X, R, or V; and

AA20 is R;

wherein X denotes a variable amino acid; W is tryptophan; C is cysteine,T is threonine, S is serine; N is asparagine; G is glycine; R isarginine; V is valine, P is proline, and Q is glutamine; and wherein thepeptide reduces blood vessel formation in a cell, tissue or organ. Invarious embodiments, the peptide contains an amino acid sequenceselected from the group consisting of:

(SEQ ID NO: 163) W-X₂-C-(T/S/N)-X₂-C-X₂-G; (SEQ ID NO: 164)W-X₂-C-S-X₂-C-G-X-G-X₃-R-X₃; (SEQ ID NO: 165)W-X₂-C-S-X₂-C-G-X₁-G-X₃-R-X₁-(R/V); (SEQ ID NO: 166)W-X₂-C-S-X-(S/R/T)-C-G-X-G-X₃-R-X-(R/V)-X; (SEQ ID NO: 167)W-X₂-C-(T/S/N)-X₂-C-X₂-G-X₅-(R/V); (SEQ ID NO: 168) P-W-X₂-C-X₃-C-X₂-G;and (SEQ ID NO: 169) (S/G/Q)-P-W-X₂-C-(T/S)-X₂-C-(G/S)-X₁-G-X₃-(R/S).

In another aspect, the invention features an isolated peptide or analogthereof containing a thrombospondin-1 domain, wherein the peptidereduces blood vessel formation in a cell, tissue or organ and contains asequence having at least 75%, 80%, 85%, 90%, 95% or 100% amino acidsequence identity to an amino acid sequence selected from peptidescontaining a TSP-1 domain listed in Table 1.

In yet another aspect, the invention features an isolated peptide oranalog thereof containing a thrombospondin-1 domain, wherein the peptidecontains a sequence having at least 75%, 80%, 85%, 90%, 95% or 100%amino acid sequence identity to an amino acid sequence selected from thegroup consisting of:

THSD-1: QPWSQCSATCGDGVRERRR; (SEQ ID NO: 64) THSD-3:SPWSPCSGNCSTGKQQRTR; (SEQ ID NO: 65) THSD-6: WTRCSSSCGRGVSVRSR;(SEQ ID NO: 66) CILP: SPWSKCSAACGQTGVQTRTR; (SEQ ID NO: 39) WISP-1:SPWSPCSTSCGLGVSTRI; (SEQ ID NO: 74) WISP-2: TAWGPCSTTCGLGMATRV;(SEQ ID NO: 75) WISP-3: TKWTPCSRTCGMGISNRV; (SEQ ID NO: 76) F-spondin:SEWSDCSVTCGKGMRTRQR; (SEQ ID NO: 73) F-spondin: WDECSATCGMGMKKRHR;(SEQ ID NO: 72) CTGF: TEWSACSKTCGMGISTRV; (SEQ ID NO: 41) fibulin-6:ASWSACSVSCGGGARQRTR; (SEQ ID NO: 45) fibulin-6: QPWGTCSESCGKGTQTRAR;(SEQ ID NO: 44) fibulin-6: SAWRACSVTCGKGIQKRSR; (SEQ ID NO: 43) CYR61:TSWSQCSKTCGTGISTRV; (SEQ ID NO: 42) NOVH: TEWTACSKSCGMGFSTRV;(SEQ ID NO: 46) UNC5-C: TEWSVCNSRCGRGYQKRTR; (SEQ ID NO: 70) UNC5-D:TEWSACNVRCGRGWQKRSR; (SEQ ID NO: 71) SCO-spondin: GPWEDCSVSCGGGEQLRSR;(SEQ ID NO: 63) Properdin: GPWEPCSVTCSKGTRTRRR; (SEQ ID NO: 49) C6:TQWTSCSKTCNSGTQSRHR; (SEQ ID NO: 38) ADAMTS-like-4:SPWSQCSVRCGRGQRSRQVR; (SEQ ID NO: 69) ADAMTS-4: GPWGDCSRTCGGGVQFSSR;(SEQ ID NO: 7) ADAMTS-8: GPWGECSRTCGGGVQFSHR; (SEQ ID NO: 14) ADAMTS-16:SPWSQCTASCGGGVQTR; (SEQ ID NO: 24) ADAMTS-18: SKWSECSRTCGGGVKFQER;(SEQ ID NO: 29) semaphorin 5A: GPWERCTAQCGGGIQARRR; (SEQ ID NO: 60)semaphorin 5A: SPWTKCSATCGGGHYMRTR; (SEQ ID NO: 61) semaphoring 5B:TSWSPCSASCGGGHYQRTR; (SEQ ID NO: 62) papilin: GPWAPCSASCGGGSQSRS;(SEQ ID NO: 48) papilin: SQWSPCSRTCGGGVSFRER; (SEQ ID NO: 47) ADAM-9:KCHGHGVCNS; (SEQ ID NO: 157) and ADAM-12: MQCHGRGVCNNRKN, (SEQ ID NO: 2)wherein A is alanine; I is isoleucine; M is methionine; H is histidine;Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T isthreonine, S is serine; N is asparagine; G is glycine; R is arginine; Vis valine, P is proline, and Q is glutamine wherein the peptide reducesblood vessel formation in a cell, tissue or organ.

In another aspect, the invention features an isolated peptide or analogthereof containing the amino acid sequence G-X₃—C-L-X—P—X₁₀—K—X-L (SEQID NO: 170), wherein the peptide reduces blood vessel formation in acell, tissue or organ.

In another aspect, the invention features an isolated peptide or analogthereof containing a 22 amino acid sequence having positions AA1-AA22(SEQ ID NO: 171), wherein:

AA1 is X, N or D; AA2 is G; AA3 is X, R or K; AA4 is X, K, E, or Q; AA5is X, A, I, L, or V; AA6 is C; AA7 is L; AA8 is X, D or N; AA9 is P;AA10 is X, A, E, D, or K; AA11 is X, A, S, or E; AA12 is P; AA13 is X,F, I, M, R, or W; AA14 is X, V, L, or I; AA15 is X, K or Q; AA16 is X, Kor R; AA17 is X, I or V; AA18 is X, I or V; AA19 is X, E or Q; AA20 isK; AA21 is X, I, F, K, or M; and AA22 is L;

wherein X denotes a variable amino acid; A is alanine; I is isoleucine;F is phenylalanine; D is aspartic acid; M is methionine; H is histidine;Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T isthreonine, S is serine; N is asparagine; G is glycine; R is arginine; Vis valine, P is proline, and Q is glutamine; and wherein the peptidereduces blood vessel formation in a cell, tissue or organ. In variousembodiments, the peptide contains an amino acid sequence selected fromthe group consisting of:

(SEQ ID NO: 170) G-X₃-C-L-X-P-X₁₀-K-X-L; (SEQ ID NO: 172)(N/D/K)-G-X₃-C-L-(D/N)-(P/L)-X₅-(K/Q)-(K/R/N)-(I/V/L)-(I/V/L)-X₆; and(SEQ ID NO: 173)(N/D)-G-(R/K)-X₂-C-L-(N/D)-P-X₂-(P/N)-X₂-(K/Q)-(K/Q)-(I/V)-(I/V)-(E/Q)-K-X-L.

In another aspect, the invention features an isolated peptide or analogthereof containing at least a fragment of a C—X—C polypeptide, whereinthe peptide contains a sequence that has at least 75%, 80%, 85%, 90%,95% or 100% amino acid sequence identity to an amino acid sequenceselected from a peptide that contains the C—X—C motif listed in Table 1.

In another aspect, the invention features an isolated peptide or analogthereof having at least 75%, 80%, 85%, 90%, 95% or 100% identity to anamino acid sequence selected from the group consisting of:

ENA-78: NGKEICLDPEAPFLKKVIQKILD; (SEQ ID NO: 95) CXCL6:NGKQVCLDPEAPFLKKVIQKILDS; (SEQ ID NO: 98) CXCL1:NGRKACLNPASPIVKKIIEKMLNS; (SEQ ID NO: 102) Gro-γ:NGKKACLNPASPMVQKIIEKIL; (SEQ ID NO: 106) Beta thromboglobulin/CXCL7:DGRKICLDPDAPRIKKIVQKKL, (SEQ ID NO: 114) Interleukin 8 (IL-8)/CXCL8:DGRELCLDPKENWVQRVVEKFLK, (SEQ ID NO: 110) GCP-2:NGKQVCLDPEAPFLKKVIQKILDS, (SEQ ID NO: 98)wherein A is alanine; I is isoleucine; F is phenylalanine; D is asparticacid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W istryptophan; C is cysteine, T is threonine, S is serine; N is asparagine;G is glycine; R is arginine; V is valine, P is proline, and Q isglutamine; and wherein the peptide reduces blood vessel formation in atissue or organ. In one embodiment, the peptide contains at least afragment of a C—X—C polypeptide.

In another aspect, the invention features an isolated peptide or analogthereof containing the amino acid sequence C—N—X₃—V—C (SEQ ID NO: 174)or P—F—X-E-C—X-G-X₅-A-N (SEQ ID NO: 175), wherein X denotes a variableamino acid; F is phenylalanine; C is cysteine, N is asparagine; G isglycine; V is valine, P is proline, and Q is glutamine wherein thepeptide reduces blood vessel formation in a tissue or organ. In oneembodiment, the peptide contains at least 5, 10, 25, 20, 25, 30, or 35amino acids of a type IV collagen polypeptide.

In yet another aspect, the invention features an isolated peptide oranalog thereof containing at least a fragment of a type IV collagen C4domain, wherein the peptide reduces blood vessel formation in a tissueor organ and contains a sequence having at least 75%, 80%, 85%, 90%, 95%or 100% identity to an amino acid sequence selected from type IVcollagen peptides listed in Table 1.

In yet another aspect, the invention features an isolated peptide oranalog thereof containing one of the following amino acid sequences:

C—N—X₃—V—C—X₂-A-X—R—N-D-X—S—Y—W-L (SEQ ID NO: 176); orL-X₂—F—S-T-X—P—F—X₂—C—N—X₃—V—C (SEQ ID NO: 177), wherein the peptidereduces blood vessel formation in a cell, tissue or organ. In oneembodiment, the sequence C—N—X₃—V—C (SEQ ID NO: 174) is 5′ of thesequence C—N—X₃—V—C—X₂-A-X—R—N-D-X—S—Y—W-L (SEQ ID NO: 176). In anotherembodiment, the sequence C—N—X₃—V—C (SEQ ID NO: 174) is 3′ of the aminoacid sequence L-X₂—F—S-T-X—P—F—X₂—C—N—X₃—V—C (SEQ ID NO: 177).

In yet another aspect, the invention features an isolated peptide oranalog thereof containing the amino acid sequenceP—F—(I/L)-E-C—X-G-X—(R/G)-X—(Y/F)—(Y/F)-A-N (SEQ ID NO: 178), whereinthe peptide reduces blood vessel formation in a cell, tissue or organ.

In yet another aspect, the invention features an isolated peptide oranalog thereof having at least 75%, 80%, 85%, 90%, 95% or 100% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of

Alpha 6 fibril of type 4 collagen: YCNINEVCHYARRNDKSYWL; (SEQ ID NO: 93)Alpha 5 fibril of type 4 collagen: LRRFSTMPFMFCNINNVCNF; (SEQ ID NO: 89)Alpha 4 fibril of type 4 collagen: AAPFLECQGRQGTCHFFAN; (SEQ ID NO: 87)Alpha 4 fibril of type 4 collagen: LPVFSTLPFAYCNIHQVCHY; (SEQ ID NO: 85)and Alpha 4 fibril of type 4 collagen: YCNIHQVCHYAQRNDRSYWL,(SEQ ID NO: 86)wherein A is alanine; I is isoleucine; F is phenylalanine; D is asparticacid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W istryptophan; C is cysteine, T is threonine, S is serine; N is asparagine;G is glycine; R is arginine; V is valine, P is proline, and Q isglutamine wherein the peptide reduces blood vessel formation in a tissueor organ.

In yet another aspect, the invention features an isolated peptide oranalog thereof containing the amino acid sequenceE-C-L-W—X-D-X₈-G-X—Y—X₅—C (SEQ ID NO: 179), wherein the peptide reducesblood vessel formation in a cell, tissue or organ. In variousembodiments, the peptide contains or consists essentially of 23, 24, 25,30, 35, or 40 amino acids of a TIMP polypeptide. In another embodiment,the peptide contains or consists essentially of an amino acid sequencehaving at least 75%, 80%, 85%, 90%, 95% or 100% amino acid sequenceidentity to ECLWTDMLSNFGYPGYQSKHYACI (SEQ ID NO: 155), wherein thepeptide reduces blood vessel formation in a cell, tissue or organ.

In yet another aspect, the invention features an isolated peptide oranalog thereof containing at least a fragment of a TIMP, wherein thepeptide contains a sequence having at least 75%, 80%, 85%, 90%, 95% or100% amino acid sequence identity to an amino acid sequence selectedfrom TIMP peptides listed in Table 1, and wherein the peptide reducesblood vessel formation in a cell, tissue or organ.

In yet another aspect, the invention features an isolated polypeptide oranalog thereof containing at least a fragment of an amino acid sequenceselected from the group consisting of SEQ ID Nos. 1-156.

In yet another aspect, the invention features an isolated peptide oranalog thereof containing or consisting essentially of an amino acidsequence having at least 75%, 80%, 85%, 90%, 95% or 100% identity to anamino acid sequence selected from the group consisting of SEQ ID Nos.1-156. In other embodiments, the peptide has at least 90%, 95%, or 100%identity to an amino acid sequence selected from the group consisting ofSEQ ID Nos. 1-156. In other embodiments, the peptide differs in at least1, 2, 3 or 4 amino acids from the amino acid sequence of SEQ ID Nos.1-156. In one embodiment, the isolated peptide or analog of any previousaspect consists of an amino acid sequence selected from the groupconsisting of SEQ ID Nos. 1-156. In other embodiments, the peptidecontains at least one modification in an amino acid of SEQ ID Nos. 1-156(e.g., a sequence alteration, modified amino acid, post-translationalmodification) that increases protease resistance, biodistribution, ortherapeutic efficacy. In other embodiments, the peptide is cyclized orpegylated.

In yet another aspect, the invention features a peptide conjugatecontaining a peptide having an amino acid sequence selected from thegroup consisting of SEQ ID Nos. 1-156 conjugated to an agent thatspecifically binds a tumor marker or endothelial cell marker. In oneembodiment, the agent (e.g., aptamer or an antibody) targets the peptideto a tumor or an endothelial cell. In other embodiments, the aptamer orantibody specifically binds fibronectin, tenascin-C, integrin, VEGF,prostate-specific membrane antigen, CD44, or tumor endothelial marker(TEM).

In yet another aspect, the invention features an isolated nucleic acidmolecule encoding the peptide of any previous aspect.

In yet another aspect, the invention features an expression vectorcontaining the nucleic acid molecule of any previous aspect, wherein thenucleic acid molecule is positioned for expression. In one embodiment,the vector further contains a promoter suitable for expressing thenucleic acid molecule in a mammalian cell.

In yet another aspect, the invention features a host cell containing thepeptide of any previous aspect or a nucleic acid molecule encoding thepeptide. In one embodiment, the cell is a prokaryotic or eukaryotic cell(e.g., a cell in vitro or in vivo). In another embodiment, the cell is ahuman cell.

In yet another aspect, the invention features a method of reducing bloodvessel formation in a cell, tissue or organ, the method involvingcontacting an endothelial cell, or a tissue or organ containing anendothelial cell with an effective amount of a peptide of any oneprevious aspect, thereby reducing blood vessel formation in the tissueor organ.

In yet another aspect, the invention features a method of reducingendothelial cell proliferation, migration, survival, or stability in atissue or organ, the method involving contacting a cell, tissue or organcontaining an endothelial cell with an effective amount of a peptide ofany one previous aspect, thereby reducing endothelial cellproliferation, migration, survival, or stability in the tissue or organ.

In yet another aspect, the invention features a method of increasingendothelial cell death in a tissue or organ, the method involvingcontacting a tissue or organ containing an endothelial cell with aneffective amount of a peptide of any previous aspect or a peptideconjugate, thereby increasing endothelial cell death in the tissue ororgan.

In various embodiments of any previous aspect, the cell, tissue or organis selected from the group consisting of bladder, bone, brain, breast,cartilage, nervous tissue, esophagus, fallopian tube, heart, pancreas,intestines, gallbladder, kidney, liver, lung, ovaries, prostate,skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus,thyroid, trachea, urogenital tract, ureter, urethra, uterus, eye, bloodcells, bone marrow, neoplastic tissue, an engineered tissue, and lymphvessels.

In yet another aspect, the invention features a method of reducing bloodvessel formation in a tissue or organ the method involving: contactingthe tissue, or organ with a vector encoding a polypeptide of SEQ ID Nos.1-156; and expressing the polypeptide in a cell of the tissue or organ,thereby reducing blood vessel formation in the tissue or organ.

In one embodiment of any previous aspect, the tissue or organ is invitro or in vivo. In another embodiment, the cell is a human cell,tissue, or organ. In yet another embodiment, the number or volume ofblood vessels in the tissue or organ are reduced by at least 10%relative to a control condition. In yet another embodiment, the peptideacts on an endothelial cell (e.g., blood vascular endothelial cells,lymph vascular endothelial cells, endothelial cell lines, primaryculture endothelial cells, endothelial cells derived from stem cells,bone marrow derived stem cells, cord blood derived cells, HUVEC,lymphatic endothelial cells, and endothelial progenitor cells).

In yet another aspect, the invention features a method for decreasingblood vessel formation in a subject in need thereof, the methodinvolving administering an effective amount of a peptide of any previousaspect or a peptide conjugate to the subject thereby decreasing bloodvessel formation.

In yet another aspect, the invention features a method of reducingendothelial cell proliferation, migration, survival, or stability in asubject in need thereof, the method involving administering an effectiveamount of a peptide of any one of claims 1-28 or a peptide conjugate tothe subject thereby reducing endothelial cell proliferation, migration,survival, or stability in the tissue or organ.

In yet another aspect, the invention features a method of increasingendothelial cell death in a subject in need thereof, the methodinvolving administering an effective amount of a peptide of any previousaspect or a peptide conjugate to the subject, thereby increasingendothelial cell death in the subject. In one embodiment, the subjecthas or is at risk of developing a disease or disorder characterized byexcess or undesirable angiogenesis or vasculogenesis. In anotherembodiment, the method ameliorates or prevents a disease or disordercharacterized by excess or undesirable angiogenesis or vasculogenesis.In still other embodiments of a previous aspect, the disease or disorderis an ocular disease selected from the group consisting of ischemicretinopathy, intraocular neovascularization, age-related maculardegeneration, corneal neovascularization, retinal neovascularization,choroidal neovascularization, diabetic macular edema, diabetic retinalischemia, diabetic retinal edema, proliferative diabetic retinopathy,retinopathy of prematurity; and persistent hyperplastic retinalsyndrome. In still other embodiments of a previous aspect, wherein thedisease or disorder is related to an undesirable immune response and isselected from the group consisting of scleroderma, psoriasis, rheumatoidarthritis, and Crohn's disease. In still other embodiments of a previousaspect, wherein the disease or disorder is a neoplasia (e.g., breastcancer, leukemia, lymphoma, solid tumor, small cell lung carcinoma,melanoma, and prostate cancer). In still other embodiments of a previousaspect the disease or disorder is an inflammatory disorder selected fromthe group consisting of asthma, osteoarthritis, chronic obstructive andpulmonary disease. In still other embodiments of a previous aspect, thedisease or disorder is a viral or bacterial infection. In still otherembodiments of a previous aspect, the excess angiogenesis is associatedwith a urogenital disorder selected from the group consisting ofendometriosis, dysfunctional uterine bleeding, and follicular cysts. Instill other embodiments, the excess angiogenesis is associated with acondition selected from the group consisting of Kaposi's sarcoma, plaqueneovascularization, atherosclerosis, restenosis, coronary collaterals,cerebral collaterals, arteriovenous malformations, angiofibroma, woundgranulation, transplant arteriopathy and atherosclerosis, vascularmalformations, DiGeorge syndrome, hereditary hemorrhagic telangiectasia,cavernous hemangioma, cutaneous hemangioma, and lymphatic malformations.In still other embodiments of a previous aspect, the excess angiogenesisis associated with radiotherapy-induced injury and chemotherapy-inducedinjury. In still other embodiments of a previous aspect, the disease ordisorder is obesity (e.g., reduces the survival or proliferation of anadipose cell or tissue). In still other embodiments, the method reducesangiogenesis or vasculogenesis or modulates blood vessel sprouting,endothelial cell proliferation, blood vessel remodeling, restenosis, orblood vessel differentiation.

In another aspect, the invention features a composition containing aneffective amount of an isolated peptide of any previous aspect or apeptide conjugate in a pharmacologically acceptable excipient.

In yet another aspect, the invention features a pharmaceuticalcomposition containing an effective amount of a nucleic acid molecule orportion thereof encoding a peptide of any previous aspect or a peptideconjugate in a pharmacologically acceptable excipient. In still otherembodiments, the composition contains 2, 3, 4, 5, 10, 15, 20, 25, 50,100, 125, 150 or more peptides of SEQ ID Nos. 1-156.

In yet another aspect, the invention features a method for identifyingan amino acid sequence of interest, the method involving:

(a) identifying an initial polypeptide or fragment thereof having aminoacid sequence identity to a reference sequence having a biologicalfunction of interest;

(b) generating a random sequence containing the amino acid compositionof the initial polypeptide of interest and comparing the random sequenceto the reference sequence to determine amino acid sequence identity,wherein said amino acid sequence identity determines a random sequencecut-off value;

(c) comparing the sequence identity of step a to the random sequencecut-off value of step b, wherein a sequence identity that issignificantly greater (5%, 10%, 20%, 25%, 50%, 75%, or 100%) than therandom sequence cut-off value identifies an amino acid sequence ofinterest. In one embodiment, the reference sequence is a thrombospondincontaining protein, collagen, CXC chemokine, kringle containing protein,somatotropin, or TIMP polypeptide.

In yet another aspect, the invention features a method for identifying apeptide having angiogenic modulating activity, the method involving:

(a) identifying a polypeptide or fragment thereof having amino acidsequence identity to a reference sequence having angiogenic modulatingactivity;

(b) identifying a hydrophobic region within the polypeptide; and

(c) comparing the amino acid sequence of the hydrophobic region with acorresponding amino acid sequence of a second organism to identify aregion having at least 75%, 80%, 85%, 90%, 95% or 100% homology, therebyidentifying a peptide having angiogenic modulating activity. In oneembodiment, the peptide inhibits or enhances angiogenesis.

In yet another aspect, the invention features a kit containing aneffective amount of a peptide selected from the group consisting of SEQID Nos. 1-156 and directions for using the peptide to treat a diseasecharacterized by undesirable or excess angiogenesis.

In yet another aspect, the invention features a method for identifyingan agent having angiogenic modulating activity, the method involvingcontacting a peptide selected from the group consisting of SEQ ID Nos.1-156 with an agent; and identifying binding of the agent to thepeptide, wherein an agent that specifically binds the peptide isidentified as having angiogenic modulating activity. In one embodiment,the agent binds the peptide.

The invention provides agents that modulate angiogenesis. Other featuresand advantages of the invention will be apparent from the detaileddescription, and from the claims.

DEFINITIONS

By “thrombospondin containing protein” is meant a protein, analog, orfragment thereof comprising at least the amino acid motifW—X₂—C—X₃—C—X₂-G (SEQ ID NO: 161), or having at least 85% amino acidsequence identity to a type 1 Thrombospondin conserved domain, andhaving angiogenesis modulating activity. Exemplary thrombospondincontaining proteins include ADAMTS 1-18, BAI 1-3, C6, CILP, Fibulin-6,papilin, properdin, semaphorin 5A, semaphorin 5B, ADAM-9, and ADAM-12.

By “Tsp-1 domain” is meant a domain containing about 50 to 61 aminoacids having homology to a domain of human thrombospondin comprisingamino acids 361-412, 417-473, or 474-530 as described by Lawler andHynes J. Cell Biol. 103:1635-1648, 1986. In particular, a Tsp-1 domainincludes 57 amino acids, including two or three conserved tryptophanresidues separated by two to four amino acids each; six conservedcysteine residues; and two highly conserved arginines and two glycinesas described by Lawler and Hynes J. Cell Biol. 103:1635-1648, 1986,which is incorporated herein by reference in its entirety.

By “Tsp-2 domain” is meant a domain containing about 55 to 61 aminoacids having homology to a domain of human thrombospondin comprisingamino acids 531-571, 572-629, or 630 to 674 as described by Lawler andHynes J. Cell Biol. 103:1635-1648, 1986. In particular, a Tsp-2 domainincludes six conserved cysteine residues and has 20% to 35% pairwiseidentity over 46 residues as described by Lawler and Hynes J. Cell Biol.103:1635-1648, 1986.

By “collagen” is meant a protein, analog, or fragment thereof comprisingat least the amino acid motif C—N—X₃—V—C (SEQ ID NO: 174), or having atleast 85% amino acid sequence identity to an α1, 2, 4, 5, or 6 domain ofcollagen IV and having angiogenesis modulating activity.

By “C—X—C chemokine” is meant a polypeptide comprising at least theamino acid motif G-X₃—C-L-X—P—X₁₀—K—X-L (SEQ ID NO: 170), or having atleast 85% amino acid sequence identity to a C—X—C chemokine comprisingthe C—X—C chemokine motif. Glu-Leu-Arg (ELR) and having angiogenesismodulating activity. Exemplary C—X—C chemokines include Gro-α/CXC1,Gro-β/CXCL2, Gro-γ/CXCL3, PF-4/CXCL4, ENA-78/CXCL5, GCP-2/CXCL6,THBG-β/CXCL7, IL-8/CXCL8, and IP-10/CXCL10.

By “kringle containing protein” is meant a polypeptide, analog, orfragment thereof comprising a kringle domain and having angiogenesismodulating activity. A kringle domain is a protein structure comprising˜80 amino acids with conserved triple disulfide bonds as defined byVaradi, A. (1984) FEBS Lett. 171, 131-136). Examplary kringle containingproteins include kininogen, AK-38 protein, lipoprotein and thrombin.

By “somatotropin” is meant a polypeptide, analog, or fragment thereofhaving at least 85% amino acid sequence identity to a member of thehuman prolactin/growth hormone family that includes a somatotropindomain See, for example, Struman Proc Natl Acad Sci USA. 1999 February16; 96(4): 1246-1251. Examplary somatotoropins include Growth Hormone-1(GH-1), Growth Hormone-2 (GH-2), somatoliberin and placental lactogen.

By “TIMP” is meant a polypeptide, analog, or fragment thereof comprisingat least the motif E-C-L-W—X-D-X₈-G-X—Y—X₅—C (SEQ ID NO: 179) or havingat least 85% amino acid sequence identity to the loop-6 domain ofTIMP-2, and having angiogenesis modulating activity. Examplary TIMPsinclude TIMP3 and TIMP4.

By “blood vessel formation” is meant the dynamic process that includesone or more steps of blood vessel development and/or maturation, such asangiogenesis, vasculogenesis, formation of an immature blood vesselnetwork, blood vessel remodeling, blood vessel stabilization, bloodvessel maturation, blood vessel differentiation, or establishment of afunctional blood vessel network.

By “angiogenesis” is meant the growth of new blood vessels originatingfrom existing blood vessels. Angiogenesis can be assayed by measuringthe total length of blood vessel segments per unit area, the functionalvascular density (total length of perfused blood vessel per unit area),or the vessel volume density (total of blood vessel volume per unitvolume of tissue).

By “vasculogenesis” is meant the development of new blood vesselsoriginating from stem cells, angioblasts, or other precursor cells.

By “blood vessel stability” is meant the maintenance of a blood vesselnetwork.

By “alteration” is meant a change in the sequence or in a modification(e.g., a post-translational modification) of a gene or polypeptiderelative to an endogeneous wild-type reference sequence.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “antibody” is meant any immunoglobulin polypeptide, or fragmentthereof, having immunogen binding ability.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

A “cancer” in an animal refers to the presence of cells possessingcharacteristics typical of cancer-causing cells, for example,uncontrolled proliferation, loss of specialized functions, immortality,significant metastatic potential, significant increase in anti-apoptoticactivity, rapid growth and proliferation rate, and certaincharacteristic morphology and cellular markers. In some circumstances,cancer cells will be in the form of a tumor; such cells may existlocally within an animal, or circulate in the blood stream asindependent cells, for example, leukemic cells.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.

By “an effective amount” is meant the amount required to ameliorate thesymptoms of a disease relative to an untreated patient. The effectiveamount of active compound(s) used to practice the present invention fortherapeutic treatment of an angiogenesis-associated disease variesdepending upon the manner of administration, the age, body weight, andgeneral health of the subject. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. Suchamount is referred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

By “isolated nucleic acid molecule” is meant a nucleic acid (e.g., aDNA) that is free of the genes, which, in the naturally occurring genomeof the organism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule which is transcribed from a DNA molecule, aswell as a recombinant DNA which is part of a hybrid gene encodingadditional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a polypeptide of the invention. An isolated polypeptideof the invention may be obtained, for example, by extraction from anatural source, by expression of a recombinant nucleic acid encodingsuch a polypeptide; or by chemically synthesizing the protein. Puritycan be measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

“By “neoplasia” is meant a disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. Solid tumors, hematological disorders, andcancers are examples of neoplasias.

By “operably linked” is meant that a first polynucleotide is positionedadjacent to a second polynucleotide that directs transcription of thefirst polynucleotide when appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the second polynucleotide.

By “peptide” is meant any fragment of a polypeptide. Typically peptidelengths vary between 5 and 1000 amino acids (e.g., 5, 10, 15, 20, 25,50, 100, 200, 250, 500, 750, and 1000).

By “polypeptide” is meant any chain of amino acids, regardless of lengthor post-translational modification.

By “promoter” is meant a polynucleotide sufficient to directtranscription.

By “reduce” is meant a decrease in a parameter (e.g., blood vesselformation) as detected by standard art known methods, such as thosedescribed herein. As used herein, reduce includes a 10% change,preferably a 25% change, more preferably a 40% change, and even morepreferably a 50% or greater change.

By “reference” is meant a standard or control condition.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and even more preferably 90%, 95%or even 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

“Sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences includes reference to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified comparison window, and can take into considerationadditions, deletions and substitutions. When percentage of sequenceidentity is used in reference to proteins it is recognized that residuepositions which are not identical often differ by conservative aminoacid substitutions, where amino acid residues are substituted for otheramino acid residues with similar chemical properties (for example,charge or hydrophobicity) and therefore do not deleteriously change thefunctional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have sequence similarity. Approaches for making thisadjustment are well-known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, for example,according to the algorithm of Meyers and Miller, Computer Applic. Biol.Sci., 4: 11-17, 1988, for example, as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

“Percentage of sequence identity” means the value determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions, substitutions, or deletions (i.e., gaps)as compared to the reference sequence (which does not compriseadditions, substitutions, or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity.

The term “substantial identity” or “homologous” in their variousgrammatical forms in the context of polynucleotides means that apolynucleotide comprises a sequence that has a desired identity, forexample, at least 60% identity, preferably at least 70% sequenceidentity, more preferably at least 80%, still more preferably at least90% and even more preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, more preferably atleast 70%, 80%, 85%, 90%, and even more preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.However, nucleic acids which do not hybridize to each other understringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This mayoccur, for example, when a copy of a nucleic acid is created using themaximum codon degeneracy permitted by the genetic code. One indicationthat two nucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid,although such cross-reactivity is not required for two polypeptides tobe deemed substantially identical.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, bearing a series of specified nucleicacid elements that enable transcription of a particular gene in a hostcell. Typically, gene expression is placed under the control of certainregulatory elements, including constitutive or inducible promoters,tissue-preferred regulatory elements, and enhancers.

A “recombinant host” may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

The term “operably linked” is used to describe the connection betweenregulatory elements and a gene or its coding region. That is, geneexpression is typically placed under the control of certain regulatoryelements, including constitutive or inducible promoters, tissue-specificregulatory elements, and enhancers. Such a gene or coding region is saidto be “operably linked to” or “operatively linked to” or “operablyassociated with” the regulatory elements, meaning that the gene orcoding region is controlled or influenced by the regulatory element.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 5, 10, or 15 amino acids,preferably at least about 20 amino acids, more preferably at least about25 amino acids, and even more preferably about 35 amino acids, about 50amino acids, about 100 amino acids, or about 150 amino acids. Fornucleic acids, the length of the reference nucleic acid sequence willgenerally be at least about 50 nucleotides, preferably at least about 60nucleotides, more preferably at least about 75 nucleotides, and evenmore preferably about 100 nucleotides about 300 nucleotides or about 450nucleotides or any integer thereabout or therebetween.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482, 1981; by the homology alignment algorithm of Needleman and Wunsch,J. Mol. Biol., 48: 443, 1970; by the search for similarity method ofPearson and Lipman, Proc. Natl. Acad. Sci. USA, 8: 2444, 1988; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 7 Science Dr.,Madison, Wis., USA; the CLUSTAL program is well described by Higgins andSharp, Gene, 73: 237-244, 1988; Corpet, et al., Nucleic Acids Research,16:881-90, 1988; Huang, et al., Computer Applications in theBiosciences, 8:1-6, 1992; and Pearson, et al., Methods in MolecularBiology, 24:7-331, 1994. The BLAST family of programs which can be usedfor database similarity searches includes: BLASTN for nucleotide querysequences against nucleotide database sequences; BLASTX for nucleotidequery sequences against protein database sequences; BLASTP for proteinquery sequences against protein database sequences; TBLASTN for proteinquery sequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York, 1995. Newversions of the above programs or new programs altogether willundoubtedly become available in the future, and can be used with thepresent invention.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite ofprograms, or their successors, using default parameters (Altschul etal., Nucleic Acids Res, 2:3389-3402, 1997). It is to be understood thatdefault settings of these parameters can be readily changed as needed inthe future.

As those ordinary skilled in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences which may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, Comput. Chem., 17:149-163, 1993) and XNU (Clayerie andStates, Comput. Chem., 17:191-1, 1993) low-complexity filters can beemployed alone or in combination.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

A “tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all precancerous andcancerous cells and tissues.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scaled similarity score (bits) for different type Ithrombospondin (TSP1) conserved domains. The similarity between thequery (vertically positioned domain) and the corresponding domains ofvarious proteins (horizontally positioned domains) is calculated.Similarities scoring higher than 45% are color coded in gray scale asshown.

FIGS. 2A-2E show scaled similarity scores (bits) for different familiesof proteins classified based on the existence of a common conserveddomain. FIG. 2A shows scores for proteins containing a CXC conserveddomain. FIG. 2B shows scores for proteins that contain a collagen typeIV domain. FIG. 2C shows scores for proteins that belong to the serpinfamily FIG. 2D shows scores for Kringle containing proteins. FIG. 2Eshows scores for proteins with a somatotropin hormone conserved domain.As in FIG. 1 the similarity between the query (vertically positioneddomain) and the corresponding domains of various proteins (horizontallypositioned domains) is calculated. Similarities scoring higher than 45%are shown in gray scale.

FIG. 3 is a graph showing an exemplary optical density reading for apeptide derived from the alpha 5 fibril of type 4 collagen applied to aHuman Umbilical Vein Endothelial Cells (HUVEC) cell culture at fourdifferent concentrations in in vitro cell proliferation assays.

FIG. 4 is a graph showing the activity of the exemplary peptide of FIG.3 applied to a HUVEC cell culture at four different concentrations in invitro cell proliferation assays; the activity is scaled using as 0% theoptical density reading from the negative control (application fullmedium only to the cell culture), and as 100% activity the opticaldensity reading from the positive control (application of full mediumand 100 ng/ml of TNP-470). All of the subsequent in vitro proliferationresults are presented in this format.

FIGS. 5A and 5B are graphs showing the activity of THSD-1 in in vitrocell proliferation assays.

FIGS. 6A and 6B are graphs showing the activity of THSD-3 in in vitrocell proliferation assays.

FIGS. 7A and 7B are graphs showing the activity of THSD-6 in in vitrocell proliferation assays.

FIGS. 8A and 8B are graphs showing the activity of CILP in in vitro cellproliferation assays.

FIGS. 9A-9F are graphs showing the activity of WISP isoforms in an invitro cell proliferation assay. FIGS. 9A and 9D show the activity of theWISP-1 derived peptide, SPWSPCSTSCGLGVSTR1 (SEQ ID NO: 74). FIGS. 9B and9E show the activity of the WISP-2 derived peptide, TAWGPCSTTCGLGMATRV(SEQ ID NO: 75). FIGS. 9C and 9F show the activity of the WISP-3 derivedpeptide TKWTPCSRTCGMGISNRV (SEQ ID NO: 76).

FIGS. 10A-10D are graphs showing the activity of two F-spondin fragmentsin in vitro cell proliferation assays.

FIGS. 11A and 11B are graphs showing the activity of CTGF in in vitrocell proliferation assays.

FIGS. 12A-12F are graphs showing the activity of three fragments offibulin-6 in an in vitro cell proliferation assay. FIGS. 12A and 12Dshows the activity profile for the ASWSACSVSCGGGARQRTR (SEQ ID NO: 45)fragment of fibulin-6; FIGS. 12B and 12E show the activity profile forQPWGTCSESCGKGTQTRAR (SEQ ID NO: 44) fragment of fibulin-6; FIGS. 12C and12E show the activity profile of the SAWRACSVTCGKGIQKRSR (SEQ ID NO: 43)fragment of fibulin-6.

FIGS. 13A and 13B are graphs showing the activity of CYR61 in in vitrocell proliferation assays.

FIGS. 14A and 14B are graphs showing the activity of NOVH after 2 and 4days of peptide application.

FIGS. 15A-15D are graphs showing the activity of two isoforms of UNC-5in an in vitro cell proliferation assay. FIGS. 15A and 15C show theactivity profile for UNC5-C TEWSVCNSRCGRGYQKRTR (SEQ ID NO: 70)fragment; FIGS. 15B and 15D show the activity profile for UNC5-DTEWSACNVRCGRGWQKRSR (SEQ ID NO: 71).

FIGS. 16A and 16B are graphs showing the activity of SCO-spondin in invitro cell proliferation assays.

FIGS. 17A and 17B are graphs showing the activity of properdin in invitro cell proliferation assays.

FIGS. 18A and 18B are graphs showing the activity of C6 in in vitro cellproliferation assays.

FIGS. 19A and 19B are graphs showing the activity of TSRC1 in in vitrocell proliferation assays.

FIGS. 20A and 20B are graphs showing the activity of ADAMTS-4 in invitro cell proliferation assays.

FIGS. 21A and 21B are graphs showing the activity of ADAMTS-8 in invitro cell proliferation assays.

FIGS. 22A and 22B are graphs showing the activity of ADAMTS-16 in invitro cell proliferation assays.

FIGS. 23 a and 23B are graphs showing the activity of ADAMTS-18 in invitro cell proliferation assays.

FIGS. 24A-24F are graphs showing the activity of Semaphorin 5A and 5Bderived peptides in in vitro cell proliferation assays. FIGS. 24A,D showthe activity profile for GPWERCTAQCGGGIQARRR (SEQ ID NO: 60) fragment ofsemaphorin 5A; FIGS. 24B,E show the activity profile forSPWTKCSATCGGGHYMRTR (SEQ ID NO: 61) fragment of semaphorin 5A; FIGS.24C,F show the activity profile for TSWSPCSASCGGGHYQRTR (SEQ ID NO: 62)fragment of semaphorin 5B.

FIGS. 25A-25D are graphs showing the activity of two different papilinfragments in in vitro cell proliferation assays. FIGS. 25A and 25C showactivity for the first fragment GPWAPCSASCGGGSQSRS (SEQ ID NO: 48);FIGS. 25B and 25D show activity for the second fragmentSPWTKCSATCGGGHYMRTR (SEQ ID NO: 61).

FIGS. 26A-26D are graphs showing the activity of fragments derived fromADAM-9 and ADAM-12 in in vitro cell proliferation assays.

FIGS. 27A and 27B are graphs showing the activity of Gro-α in in vitrocell proliferation assays.

FIGS. 28A and 28B are graphs showing the activity of Gro-γ in in vitrocell proliferation assays.

FIGS. 29A and 29B are graphs showing the activity of THBG in in vitrocell proliferation assays.

FIGS. 30A and 30B are graphs showing the activity of the IL-8 fragmentin in vitro cell proliferation assays.

FIGS. 31A and 31B are graphs showing the activity of the ENA-78 fragmentin in vitro cell proliferation assays.

FIGS. 32A and 32B are graphs showing the activity of the GCP-2 fragmentin in vitro cell proliferation assays.

FIGS. 33A and 33B are graphs showing the activity of the C4-alpha6fragment in in vitro cell proliferation assays.

FIGS. 34A-34D are graphs showing the activity of the C4-alpha5 fragmentsin in vitro cell proliferation assays.

FIGS. 35A-35F are graphs showing the activity of the C4-alpha4 fragmentsin in vitro cell proliferation assays.

FIGS. 36A and 36B are graphs showing the activity of the TIMP-3 fragmentin in vitro cell proliferation assays.

FIGS. 37A and 37B are graphs showing the activity of two scrambledpeptides in in vitro cell proliferation assays.

FIGS. 38A and 38B show in vivo angiogenesis inhibition in C57BL/6 micein an experiment using subcutaneously implantedbasement-membrane-extract filled DIVAA angioreactors.

FIG. 39 shows the 4-letter motif common in all the experimentally testedTSP-1 containing proteins. (SEQ ID NOS 7, 14, 24, 29, 38, 40-42, 72, 73,44, 43, 45-49, 63, 61, 62, 64-66, 69-71 & 74-76 are disclosedrespectively in order of appearance.)

FIG. 40 shows common motifs of the TSP-1 containing peptides using athreshold of 60% (A) (SEQ ID NOS 40-42, 73, 44, 43, 45, 46, 48, 47, 63,61, 62, 64, 69, & 74-76 are disclosed respectively in order ofappearance) and 45% (B) (SEQ ID NOS 24, 7, 14, 40, 44, 48, 49, 63, 61,64, 65, 69 & 74 are disclosed respectively in order of appearance).

FIG. 41 shows the 4-letter motif common in all the theoreticallypredicted TSP-1 containing proteins. In the shaded insert the predictedmotif is identified within TSP-2 domains as well. (SEQ ID NOS 3-16,18-33, 35-38, 40-42, 72, 73, 43-49, 63-71, & 74-76 are disclosedrespectively in order of appearance.)

FIG. 42 shows the 6-letter motif common in all the experimentally testedC—X—C containing proteins. (SEQ ID NOS 102, 106, 95, 98, 114 & 110 aredisclosed respectively in order of appearance.)

FIG. 43 shows the common motif in all the theoretically predictedanti-angiogenic C—X—C containing proteins. (SEQ ID NOS 109, 110, 95,98-101, 96, 97, 114, 116, 115, 113, 105, 108, 103, 104, 106, 107, 102 &111 are disclosed respectively in order of appearance.)

FIGS. 44A-44C show the most abundant motif in the theoreticallypredicted anti-angiogenic collagen derived peptide fragments. FIGS. 44Band 44C show novel motifs when shifting the abundant 7-mer downstream(B) or upstream (C) in the peptide sequences. (A) SEQ ID NOS 79, 90, 77,78, 89, 88, 92, 93, 81, 82 & 84-86 are disclosed respectively in orderof appearance. (B) SEQ ID NOS 77, 78, 89, 88, 92, 81, 94, & 85 aredisclosed respectively in order of appearance. (C) SEQ ID NOS 79, 90,77, 88, 92, 93, 81, 82, 85 & 86 are disclosed in order of appearance.

FIG. 45 shows a less common motif within the sequences of collagenderived peptide fragments. SEQ ID NOS 80, 91, 83, 94, & 87 are disclosedrespectively in order of appearance.

FIG. 46 shows the common motif in all the predicted anti-angiogenicfragments derived from TIMPs. SEQ ID NOS 155, 156, & 160 are disclosedrespectively in order of appearance.

FIG. 47 shows the effect of applied peptides on the migration of HUVECsusing a modified Boyden chamber. The cells were allowed to migrate for16 hours. The peptides applied were C4α6(seq.1): YCNINEVCHYARRNDKSYWL(SEQ ID NO: 93), C4α5(seq.1): LRRFSTMPFMFCNINNVCNF (SEQ ID NO: 89),TIMP3: ECLWTDMLSNFGYPGYQSKHYACI (SEQ ID NO: 155) C4α5(seq.2):FCNINNVCNFASRNDYSYWL (SEQ ID NO: 90), C4α6(seq.2): ATPFIECSGARGTCHYFAN(SEQ ID NO: 94), C4α4(seq.1): AAPFLECQGRQGTCHFFAN (SEQ ID NO: 87),C4α4(seq.2): LPVFSTLPFAYCNIHQVCHY (SEQ ID NO: 85), C4α4(seq.3):YCNIHQVCHYAQRNDRSYWL (SEQ ID NO: 86), C4α5(seq.3): SAPFIECHGRGTCNYYANS(SEQ ID NO: 91), TIMP4: ECLWTDWLLERKLYGYQAQHYVCM (SEQ ID NO: 156).

DETAILED DESCRIPTION OF THE INVENTION

The invention generally features compositions and methods that areuseful for modulating blood vessel formation, as well as methods thatprovide for the systematic and efficient identification of angiogenesisinhibitors. As described in more detail below, a systematiccomputational methodology based on bioinformatics was used to identifyand classify novel putative endogenous inhibitors of angiogenesis. Alist of proteins and protein fragments having anti-angiogenic orpro-apoptotic properties was compiled. Based on similarity to theseknown anti-angiogenic fragments and the existence of conserved domainswithin these sequences, novel putative angiogenic inhibitors wereidentified and classified. These novel angiogenic inhibitors includemembers of the ADAM, ADAMTS, CXC and semaphorin protein families, aswell as coagulation factors, receptor tyrosine kinase-like orphanreceptors and various kringle-containing proteins. Clustering ofsimilarities among the hits allowed predictions to be made concerningthe localization of the anti-angiogenic activity within the proteinsequences. The powerful computational methods used provided for theefficient identification of novel anti-angiogenic proteins andfragments. The anti-angiogenic activity of a variety of these peptideshas been characterized experimentally in vitro and/or in vivo.

Angiogenesis

Angiogenesis, which involves the growth or sprouting of new microvesselsfrom pre-existing vasculature, and vasculogenesis, which involves denovo vascular growth, is essential to many physiological andpathological conditions, including embryogenesis, cancer, rheumatoidarthritis, diabetic retinopathy, obesity, atherosclerosis, ischemicheart and limb disease, and wound healing. Over 70 diseases have beenidentified as angiogenesis dependent (Carmeliet, Nature, 438:932-6,2005). Under physiological conditions, the growth of new microvessels istightly regulated and orchestrated by maintaining a balance betweenendogenous pro- and anti-angiogenic factors. Tipping the balance of thisregulation may lead to either excessive neovascularization, as incancer, age-related macular degeneration, and rheumatoid arthritis, orinsufficient vascularization, as in ischemic heart and limb disease,ischemic brain, and neural degeneration.

Angiogenesis is a complex multistep process that involves interactionsbetween endothelial cells (EC), pericytes, vascular smooth muscle cells,and stromal cells (e.g., stem cells and parenchymal cells). Theseinteractions occur through secreted factors, such as vascularendothelial growth factor (VEGF), platelet-derived growth factor (PDGF),basic fibroblast growth factor (bFGF or FGF-2) and angiopoietins, aswell as through cell-cell and cell-extracellular matrix (ECM)interactions. Endothelial cell-ECM interactions regulate numerousprocesses that are critical for angiogenesis, including endothelial cellmigration, proliferation, differentiation and apoptosis. Angiogenicprocesses include network stabilization and remodeling that may involvethe recruitment of stromal cells, as well as the pruning of somevessels. In many cases, angiogenesis occurs as a response to hypoxia. Atranscription factor called hypoxia-inducible factor, HIF1α, has beendemonstrated to act as an oxygen sensor whose activity leads toupregulation of VEGF in parenchymal and stromal cells (Semenza,Physiology (Bethesda), 19:176-82, 2004). VEGF is secreted as a homodimerin the form of several heparin-binding and non-heparin-bindingsplice-variant isoforms; it diffuses through the interstitial space andcan bind to the endothelial cell receptors VEGFR1 and VEGFR2, as well asco-receptors such as Neuropilin-1, thus initiating a signal transductioncascade that leads to endothelial cell proliferation and migration. Theproduction of endothelial cell matrix metalloproteinases, MMPs,increases as a result of endothelial cell activation; MMPs are necessaryfor selectively clipping the capillary basement membrane and the ECM,which constitute physical barriers to endothelial cell migration andcapillary sprouting. MMPs and their associated molecules also play acrucial role in uncovering cryptic sites of the ECM proteins, a numberof which have been identified as anti-angiogenic (Davis et al., AnatRec, 268:252-75, 2002; Folkman, Annu Rev Med, 57:1-18, 2006; Rundhaug, JCell Mol Med, 9:267-85, 2005; Schenk and Quaranta, Trends Cell Biol,13:366-75, 2003), and in processing cell-surface receptors (Mott andWerb, Curr Opin Cell Biol, 16:558-64, 2004).

Diseases Associated with Undesirable Angiogenesis

Where the processes regulating angiogenesis are disrupted, pathology mayresult. Such pathology affects a wide variety of tissues and organsystems. Diseases characterized by excess or undesirable angiogenesisare susceptible to treatment with therapeutic agents described herein.

Excess angiogenesis in numerous organs is associated with cancer andmetastasis, including neoplasia and hematologic malignancies.

Angiogenesis-related diseases and disorders are commonly observed in theeye where they may result in blindness. Such disease include, but arenot limited to, age-related macular degeneration, choroidalneovascularization, persistent hyperplastic vitreous syndrome, diabeticretinopathy, and retinopathy of prematurity (ROP).

A number of angiogenesis-related diseases are associated with the bloodand lymph vessels including transplant arteriopathy and atherosclerosis,where plaques containing blood and lymph vessels form, vascularmalformations, DiGeorge syndrome, hereditary hemorrhagic telangiectasia,cavernous hemangioma, cutaneous hemangioma, and lymphatic malformations.

Other angiogenesis diseases and disorders affect the bones, joints,and/or cartilage include, but are not limited to, arthritis, synovitis,osteomyelitis, osteophyte formation, and HIV-induced bone marrowangiogenesis.

The gastro-intestinal tract is also susceptible to angiogenesis diseasesand disorders. These include, but are not limited to, inflammatory boweldisease, ascites, peritoneal adhesions, and liver cirrhosis.

Angiogenesis diseases and disorders affecting the kidney include, butare not limited to, diabetic nephropathy (early stage: enlargedglomerular vascular tufts).

Excess angiogenesis in the reproductive system is associated withendometriosis, uterine bleeding, ovarian cysts, ovarianhyperstimulation.

In the lung, excess angiogenesis is associated with primary pulmonaryhypertension, asthma, nasal polyps, rhinitis, chronic airwayinflammation, cystic fibrosis.

Diseases and disorders characterized by excessive or undesirableangiogenesis in the skin include psoriasis, warts, allergic dermatitis,scar keloids, pyogenic granulomas, blistering disease, Kaposi's sarcomain AIDS patients, systemic sclerosis.

Obesity is also associated with excess angiogenesis (e.g., angiogenesisinduced by fatty diet). Adipose tissue may be reduced by theadministration of angiogenesis inhibitors.

Excess angiogenesis is associated with a variety of auto-immunedisorders, such as systemic sclerosis, multiple sclerosis, Sjögren'sdisease (in part by activation of mast cells and leukocytes).Undesirable angiogenesis is also associated with a number of infectiousdiseases, including those associated with pathogens that express(lymph)-angiogenic genes, that induce a (lymph)-angiogenic program orthat transform endothelial cells. Such infectious disease include thosebacterial infections that increase HIF-1 levels, HIV-Tat levels,antimicrobial peptides, levels, or those associated with tissueremodeling.

Infectious diseases, such as viral infections, can cause excessiveangiogenesis which is susceptible to treatment with agents of theinvention. Examples of viruses that have been found in humans include,but are not limited to, Retroviridae (e.g. human immunodeficiencyviruses, such as HIV-1 (also referred to as HDTV-III, LAVE orHTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.strains that cause gastroenteritis); Togaviridae (e.g. equineencephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses,encephalitis viruses, yellow fever viruses); Coronoviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes virus; Poxyiridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and relatedviruses, and astroviruses).

The present invention provides methods of treating diseases and/ordisorders or symptoms thereof associated with excess or undesiredangiogenesis, which comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising a compound of theformulae herein to a subject (e.g., a mammal, such as a human). Thus,one embodiment is a method of treating a subject suffering from orsusceptible to an angiogenesis-related disease or disorder or symptomthereof. The method includes the step of administering to the mammal atherapeutic amount of an amount of a compound herein sufficient to treatthe disease or disorder or symptom thereof (e.g., to prevent or reduceangiogenesis) under conditions such that the disease or disorder istreated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein (e.g., a peptide described herein, ormimetic, or analog thereof), or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject (e.g., animal, human) in need thereof,including a mammal, particularly a human. Such treatment will besuitably administered to subjects, particularly humans, suffering from,having, susceptible to, or at risk for a disease, disorder, or symptomthereof. Determination of those subjects “at risk” can be made by anyobjective or subjective determination by a diagnostic test or opinion ofa subject or health care provider (e.g., genetic test, enzyme or proteinmarker, Marker (as defined herein), family history, and the like). Thecompounds herein may be also used in the treatment of any otherdisorders in which angiogenesis may be implicated.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., any target delineated hereinmodulated by a compound herein, a protein or indicator thereof, etc.) ordiagnostic measurement (e.g., screen, assay) in a subject suffering fromor susceptible to a disorder or symptoms thereof associated withangiogenesis, in which the subject has been administered a therapeuticamount of a compound herein sufficient to treat the disease or symptomsthereof. The level of Marker determined in the method can be compared toknown levels of Marker in either healthy normal controls or in otherafflicted patients to establish the subject's disease status. Inpreferred embodiments, a second level of Marker in the subject isdetermined at a time point later than the determination of the firstlevel, and the two levels are compared to monitor the course of diseaseor the efficacy of the therapy. In certain preferred embodiments, apre-treatment level of Marker in the subject is determined prior tobeginning treatment according to this invention; this pre-treatmentlevel of Marker can then be compared to the level of Marker in thesubject after the treatment commences, to determine the efficacy of thetreatment.

Treatment of Neoplasia

The methods of the invention are particularly well suited for thetreatment of neoplasias. By “neoplasia” is meant a disease that iscaused by or results in inappropriately high levels of cell division,inappropriately low levels of apoptosis, or both. For example, cancer isan example of a proliferative disease. Examples of cancers include,without limitation, leukemias (e.g., acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia, chronic leukemia, chronic myelocyticleukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma(Hodgkin's disease, non-Hodgkin's disease), Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors, such assarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

Peptides of the Invention

The present invention utilizes powerful computational and bioinformaticapproaches to identify therapeutic agents (e.g., polypeptides, peptides,analogs, and fragments thereof) having anti-angiogenic activity. Theamino acid sequences of such agents are provided at Table 1 (below),which provides peptide sequences of peptides of the invention, as wellas the names of the proteins from which they are derived, GenBankAccession Nos., and the amino acid positions of the sequences Aminoacids are referred to herein by their commonly known one-letter symbolsrecommended by the IUPAC-IUB Biochemical Nomenclature Commission; theycan also be referred to by their commonly known three letter symbols.

TABLE 1 Anti-Angiogenic Peptide sequences SEQ ID NO.Thrombospondin Containing Proteins   1 ADAM-9 Q13443: 649-661KCHGHGVCNSNKN   2 ADAM-12 Q43184: 662-675 MQCHGRGVCNNRKN   3 ADAMTS-1Q9UHI8: 566-584 GPWGDCSRTCGGGVQYTMR   4 ADAMTS-2 CAA05880.1: 982-998GPWSQCSVTCGNGTQER   5 ADAMTS-3 NP_055058.1: 973-989 GPWSECSVTCGEGTEVR  6 ADAMTS-4 CAH72146.1: 527-540 GPWGDCSRTCGGGV   7 ADAMTS-4CAH72146.1: 527-545 GPWGDCSRTCGGGVQFSSR   8 ADAMTS-5NP_008969.1: 882-898 GPWLACSRTCDTGWHTR   9 ADAMTS-6 NP_922932.2: 847-860QPWSECSATCAGGV  10 ADAMTS-6 NP_922932.2: 847-863 QPWSECSATCAGGVQRQ  11ADAMTS-7 AAH61631.1: 1576-1592 GPWGQCSGPCGGGVQRR  12 ADAMTS-7AAH61631.1: 828-841 GPWTKCTVTCGRGV  13 ADAMTS-8 Q9UP79: 534-547GPWGECSRTCGGGV  14 ADAMTS-8 Q9UP79: 534-552 GPWGECSRTCGGGVQFSHR  15ADAMTS-9 Q9P2N4: 1247-1261 WSSCSVTCGQGRATR  16 ADAMTS-9Q9P2N4: 1335-1351 GPWGACSSTCAGGSQRR  17 ADAMTS-9 Q9P2N4: 595-613SPFGTCSRTCGGGIKTAIR  18 ADAMTS-10 Q9H324: 528-546 TPWGDCSRTCGGGVSSSSR 19 ADAMTS-12 P58397: 1479-1493 WDLCSTSCGGGFQKR  20 ADAMTS-12P58397: 549-562 SPWSHCSRTCGAGV  21 ADAMTS-13 AAQ88485.1: 751-765WMECSVSCGDGIQRR  22 ADAMTS-14 CAI13857.1: 980-994 WSQCSATCGEGIQQR  23ADAMTS-15 CAC86014.1: 900-916 SAWSPCSKSCGRGFQRR  24 ADAMTS-16Q8TE57: 1133-1149 SPWSQCTASCGGGVQTR  25 ADAMTS-16 Q8TE57: 1133-1150SPWSQCTASCGGGVQTRS  26 ADAMTS-18 Q8TE60: 1131-1146 PWQQCTVTCGGGVQTR  27ADAMTS-18 Q8TE60: 1131-1147 PWQQCTVTCGGGVQTRS  28 ADAMTS-18Q8TE60: 998-1014 GPWSQCSKTCGRGVRKR  29 ADAMTS-18 Q8TE60: 596-614SKWSECSRTCGGGVKFQER  30 ADAMTS-19 CAC84565.1: 1096-1111 WSKCSITCGKGMQSRV 31 ADAMTS-20 CAD56159.3: 1478-1494 NSWNECSVTCGSGVQQR  32 ADAMTS-20CAD56159.3: 1309-1326 GPWGQCSSSCSGGLQHRA  33 ADAMTS-20CAD56159.3: 1661-1675 WSKCSVTCGIGIMKR  34 ADAMTS-20 CAD56160.2: 564-581PYSSCSRTCGGGIESATR  35 BAI-1 O14514: 361-379 SPWSVCSSTCGEGWQTRTR  36BAI-2 O60241: 304-322 SPWSVCSLTCGQGLQVRTR  37 BAI-3 CAI21673.1: 352-370SPWSLCSFTCGRGQRTRTR  38 C6 AAB59433.1: 30-48 TQWTSCSKTCNSGTQSRHR  39CILP AAQ89263.1: 156-175 SPWSKCSAACGQTGVQTRTR  40 CILP-2AAN17826.1: 153-171 GPWGPCSGSCGPGRRLRRR  41 CTGF CAC44023.1: 204-221TEWSACSKTCGMGISTRV  42 CYR61 AAR05446.1: 234-251 TSWSQCSKTCGTGISTRV  43Fibulin-6 CAC37630.1: 1574-1592 SAWRACSVTCGKGIQKRSR  44 Fibulin-6CAC37630.1: 1688-1706 QPWGTCSESCGKGTQTRAR  45 Fibulin-6CAC37630.1: 1745-1763 ASWSACSVSCGGGARQRTR  46 NOVH AAL92490.1: 211-228TEWTACSKSCGMGFSTRV  47 Papilin NP_775733.2: 33-51 SQWSPCSRTCGGGVSFRER 48 Papilin NP_775733.2: 342-359 GPWAPCSASCGGGSQSRS  49 ProperdinAAP43692.1: 143-161 GPWEPCSVTCSKGTRTRRR  50 ROR-1 CAH71706.1: 313-391CYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC  51 ROR-1 CAH71706.1: 310-391NHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC  52 ROR-1 CAH71706.1: 311-388HKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDI  53 ROR-1 CAH71706.1: 311-391HKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC  54 ROR-1 CAH71706.1: 312-392KCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACD  55 ROR-2 Q01974: 315-395QCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCS  56 ROR-2 Q01974: 314-391HQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDV  57 ROR-2 Q01974: 314-394HQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSC  58 ROR-2 Q01974: 314-395HQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCS  59 ROR-2 Q01974: 315-394QCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSC  60 Semaphorin 5ANP_003957.1: 660-678 GPWERCTAQCGGGIQARRR  61 Semaphorin 5ANP_003957.1: 848-866 SPWTKCSATCGGGHYMRTR  62 Semaphorin 5BAAQ88491.1: 916-934 TSWSPCSASCGGGHYQRTR  63 SCO-spondinXP_379967.2: 3781-3799 GPWEDCSVSCGGGEQLRSR  64 THSD1 AAQ88516.1: 347-365QPWSQCSATCGDGVRERRR  65 THSD3 AAH33140.1: 280-298 SPWSPCSGNCSTGKQQRTR 66 THSD6 AAH40620.1: 44-60 WTRCSSSCGRGVSVRSR  67 TSP-2CAI23645.1: 444-462 SPWSSCSVTCGVGNITRIR  68 TSP-2 CAI23645.1: 501-519SPWSACTVTCAGGIRERTR  69 TSRC1 AAH27478.1: 140-159 SPWSQCSVRCGRGQRSRQVR 70 UNCSC AAH41156.1: 267-285 TEWSVCNSRCGRGYQKRTR  71 UNCSDAAQ88514.1: 259-277 TEWSACNVRCGRGWQKRSR  72 VSGP/F-spondinBAB18461.1: 567-583 WDECSATCGMGMKKRHR  73 VSGP/F-spondinBAB18461.1: 621-639 SEWSDCSVTCGKGMRTRQR  74 WISP-1 AAH74841.1: 221-238SPWSPCSTSCGLGVSTRI  75 WISP-2 AAQ89274.1: 199-216 TAWGPCSTTCGLGMATRV  76WISP-3 CAB16556.1: 191-208 TKWTPCSRTCGMGISNRV Collagens  77 α1CIVCAH74130.1: 1479-1556 NERAHGQDLGTAGSCLRKFSTMPFLFCNINNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAM  78 α1CIV CAH74130.1: 1494-1513LRKFSTMPFLFCNINNVCNF  79 α1CIV CAH74130.1: 1504-1523FCNINNVCNFASRNDYSYWL  80 α1CIV CAH74130.1: 1610-1628 SAPFIECHGRGTCNYYANA 81 α2CIV CAH71366.1: 1517-1593QEKAHNQDLGLAGSCLARFSTMPFLYCNPGDVCYYASRNDKSYWLSTTAPLPMMPVAEDEIKPYISRCSVCEAPAIA  82 α2CIV CAH71366.1: 1542-1561YCNPGDVCYYASRNDKSYWL  83 α2CIV CAH71366.1: 1646-1664 ATPFIECNGGRGTCHYYAN 84 α4CIV CAA56943.1: 1499-1575QEKAHNQDLGLAGSCLPVFSTLPFAYCNIHQVCHYAQRNDRSYWLASAAPLPMMPLSEEAIRPYVSRCAVCEAPAQA  85 α4CIV CAA56943.1: 1514-1533LPVFSTLPFAYCNIHQVCHY  86 α4CIV CAA56943.1: 1524-1543YCNIHQVCHYAQRNDRSYWL  87 α4CIV CAA56943.1: 1628-1646 AAPFLECQGRQGTCHFFAN 88 α5CIV AAC27816.1: 1495-1572NKRAHGQDLGTAGSCLRRFSTMPFMFCNINNVCNFASRNDYSYWLSTPEPMPMSMQPLKGQSIQPFISRCAVCEAPAV  89 α5CIV AAC27816.1: 1510-1529LRRFSTMPFMFCNINNVCNF  90 α5CIV AAC27816.1: 1520-1539FCNINNVCNFASRNDYSYWL  91 α5CIV AAC27816.1: 1626-1644 SAPFIECHGRGTCNYYANS 92 α6CIV CAI40758.1: 1501-1577QEKAHNQDLGFAGSCLPRFSTMPFIYCNINEVCHYARRNDKSYWLSTTAPIPMMPVSQTQIPQYISRCSVCEAPSQA  93 α6CIV CAI40758.1: 1526-1545YCNINEVCHYARRNDKSYWL  94 α6CIV CAI40758.1: 1630-1648 ATPFIECSGARGTCHYFANCXC Chemokines  95 ENA-78/CXCL5 AAP35453.1: 86-108NGKEICLDPEAPFLKKVIQKILD  96 ENA-78/CXCL5 AAP35453.1: 48-103RCVCLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKNGKEIC LDPEAPFLKKVI  97ENA-78/CXCL5 AAP35453.1: 51-107CLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKNGKEICLDP EAPFLKKVIQKIL  98GCP-2/CXCL6 AAH13744.1: 86-109 NGKQVCLDPEAPFLKKVIQKILDS  99 GCP-2/CXCL6AAH13744.1: 47-106 LRCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQVCLDPEAPFLKKVIQKI 100 GCP-2/CXCL6 AAH13744.1: 48-103RCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQV CLDPEAPFLKKVI 101GCP-2/CXCL6 AAH13744.1: 51-107CLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQVCLDP EAPFLKKVIQKIL 102GRO-α/CXCL1 AAP35526.1: 80-103 NGRKACLNPASPIVKKIIEKMLNS 103 GRO-α/CXCL1AAP35526.1: 42-97 RCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKII 104 GRO-α/CXCL1 AAP35526.1: 44-101QCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLN PASPIVKKIIEKML 105Gro-β/CXCL2 AAH15753.1: 42-97RCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKAC LNPASPMVKKII 106GRO-γ/MIP-2β/CXCL3 AAA63184.1: 79-100 NGKKACLNPASPMVQKIIEKIL 107GRO-γ/MIP-2β/CXCL3 AAA63184.1: 43-100QCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKACLN PASPMVQKIIEKIL 108GRO-γ/MIP-2β/CXCL3 AAA63184.1: 41-96RCQCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKAC LNPASPMVQKII 109 IL-8/CXCL8AAP35730.1: 35-94 QCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLK 110 IL-8/CXCL8 AAP35730.1: 72-94 DGRELCLDPKENWVQRVVEKFLK111 IP-10/CXCL10 AAH10954.1: 29-86RCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCL NPESKAIKNLL 112MIG/CXCL9 Q07325: 32-91 SCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEK 113 PF-4/CXCL4 AAK29643.1: 43-100CVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQ APLYKKIIKKLLE 114THBG-β/CXCL7 AAB46877.1: 100-121 DGRKICLDPDAPRIKKIVQKKL 115 THBG-β/CXCL7AAB46877.1: 62-117 RCMCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDPDAPRIKKIV 116 THBG-β/CXCL7 AAB46877.1: 64-121MCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDP DAPRIKKIVQKKLKringle Containing Proteins 117 AK-38 protein AAK74187.1: 14-93DCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCA 118 AK-38 proteinAAK74187.1: 12-94 QDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCA 119 AK-38 proteinAAK74187.1: 13-90 DCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDI 120 AK-38 protein AAK74187.1: 14-93CMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLC 121 Hageman fct/cf AAM97932.1: 216-292 SCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQARN XIIWGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDL 122 Hageman fct/cf AAM97932.1: 214-295 KASCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQAR XIINWGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDLAQC 123 Hageman fct/cf AAM97932.1: 215-296 ASCYDGRGLSYRGLARTTLSGAPCQPWASEATYRNVTAEQARN XIIWGLGGHAFCRNPDNDIRPWCFVLNRDRLSWEYCDLAQCQ 124 HGF P14210: 127-206NCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQC 125 HGF P14210: 127-207NCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCS 126 HGF P14210: 304-377ECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYC 127 HGF P14210: 210-289ECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCA 128 HGF P14210: 304-383ECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNC 129 HyaluronanNP_004123.1: 192-277 DDCYVGDGYSYRGKMNRTVNQHACLYWNSHLLLQENYNMFM bindingEDAETHGIGEHNFCRNPDADEKPWCFIKVTNDKVKWEYCDVSA CS 130 HyaluronanNP_004123.1: 192-276 DDCYVGDGYSYRGKMNRTVNQHACLYWNSHLLLQENYNMFM bindingEDAETHGIGEHNFCRNPDADEKPWCFIKVTNDKVKWEYCDVSA C 131 KREMEN-1BAB40969.1: 31-114 ECFTANGADYRGTQNWTALQGGKPCLFWNETFQHPYNTLKYPNGEGGLGEHNYCRNPDGDVS- PWCYVAEHEDGVYWKYCEIPAC 132 KREMEN-1BAB40969.1: 31-115 ECFTANGADYRGTQNWTALQGGKPCLFWNETFQHPYNTLKYPNGEGGLGEHNYCRNPDGDVSPWCYVAEHEDGVYWKYCEIPAC Q 133 KREMEN-2BAD97142.1: 35-119 ECFQVNGADYRGHQNRTGPRGAGRPCLFWDQTQQHSYSSASDPHGRWGLGAHNFCRNPDGDVQ-PWCYVAETEEGIYWRYCDIPSC 134 KREMEN-2BAD97142.1: 34-119 SECFQVNGADYRGHQNRTGPRGAGRPCLFWDQTQQHSYSSASDPHGRWGLGAHNFCRNPDGDVQPWCYVAETEEGIYWRYCDIPSC 135 Lp(a)NP_005568.1: 1615-1690 TEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPG 136 Lp(a) NP_005568.1: 3560-3639QDCYYHYGQSYRGTYSTTVTGRTCQAWSSMTPHQHSRTPENYPNAGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTQC 137 Lp(a) NP_005568.1: 4123-4201QCYHGNGQSYRGTFSTTVTGRTCQSWSSMTPHRHQRTPENYPNDGLTMNYCRNPDADTGPWCFTMDPSIRWEYCNLTRC 138 Lp(a) NP_005568.1: 4225-4308EQDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCA 139 MacrophageAAH48330.1: 188-268 EAACVWCNGEEYRGAVDRTESGRECQRWDLQHPHQHPFEPGK stim. 1FLDQGLDDNYCRNPDGSERPWCYTTDPQIEREFCDLPRC 140 MacrophageAAH48330.1: 368-448 QDCYHGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEP stim. 1HAQLEENFCRNPDGDSHGPWCYTMDPRTPFDYCALRRC 141 MacrophageAAH48330.1: 368-449 QDCYHGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEP stim. 1HAQLEENFCRNPDGDSHGPWCYTMDPRTPFDYCALRRCA 142 MacrophageAAH48330.1: 370-448 CYHGAGEQYRGTVSKTRKGVQCQRWSAETPHKPQFTFTSEPHA stim. 1QLEENFCRNPDGDSHGPWCYTMDPRTPFDYCALRRC 143 Thrombin/cf IIAAL77436.1: 105-186 EGNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVC 144 Thrombin/cf IIAAL77436.1: 106-186 GNCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVC 145 Thrombin/cf IIAAL77436.1: 107-183 NCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSI 146 Thrombin/cf II AAL77436.1: 107-186NCAEGLGTNYRGHVNITRSGIECQLWRSRYPHKPEINSTTHPGADLQENFCRNPDSSTTGPWCYTTDPTVRRQECSIPVC 147 tPA AAH95403.1: 214-293DCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDV 148 tPA AAH95403.1: 213-296SDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDVPSC 149 tPA AAH95403.1: 213-297SDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDVPSCS 150 tPA AAH95403.1: 214-296DCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKSRRLTWEYCDVPSC Somatotropins 151 GH-1NP_000506.2: 26-160 AFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPR 152 GH-2 CAG46700.1: 26-160AFPTIPLSRLFDNAMLRARRLYQLAYDTYQEFEEAYILKEQKYSFLQNPQTSLCFSESIPTPSNRAKTQQKSNLELLRISLLLIQSWLEPVQLLRSVFANSLVYGASDSNVYRHLKDLEEGIQTLMWRLEDGSP R 153 PlacentalAAP35572.1: 26-160 AVQTVPLSRLFDHAMLQAHRAHQLAIDTYQEFEETYIPKDQKYS lactogenFLHDSQTSFCFSDSIPTPSNMEETQQKSNLELLRISLLLIESWLEPVRFLRSMFANNLVYDTSDSDDYHLLKDLEEGIQTLMGRLEDGSR R 154 SomatoliberinAAH62475.1: 26-145 AFPTIPLSRLFDNASLRAHRLHQLAFDTYQEFNPQTSLCFSESIPTPSMREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPR TIMPs 155 TIMP 3 AAA21815.1: 148-171ECLWTDMLSNFGYPGYQSKHYACI 156 TIMP 4 AAV38433.1: 175-198ECLWTDWLLERKLYGYQAQHYVCM

Angiogenesis Assays

The biological activity of therapeutic agents of the invention ischaracterized using any method for assaying angiogenic activity known inthe art. In vitro angiogenesis assays have been described in detail inrecent reviews (Akhtar et al., Angiogenesis, 5:75-80, 2002; Auerbach etal., Cancer Metastasis Rev, 19:167-72, 2000; Auerbach et al., Clin Chem,49:32-40, 2003; Staton et al., Int J Exp Pathol, 85:233-48, 2004; Vailheet al., Lab Invest, 81:439-52, 2001). There are a number of differentendothelial cell lineages that have been used in angiogenesis assays:bovine aortic, bovine retinal, rat and mouse microvascular, humanaortic, human bladder microvascular, human cardiac microvascular, humandermal microvascular, human lung microvascular and human umbilical veinendothelial cells. All of these endothelial cells are capable ofdifferentiating in vitro and forming capillary-like structures. Thisprocess occurs when the cells are cultured in a monolayer ofextracellular matrix components, such as the Matrigel (extracellularmatrix material similar to basement membrane), type I collagen,fibronectin or laminin. An endothelial cell lineage that is commonlyused for testing the angiogenic response is the human umbilical veinendothelial cells (HUVECs). The National Cancer Institute (NCI) hasissued guidelines for testing the anti-angiogenic efficacy of novelagents (http://dtp.nci.nih.gov/aa-resources/aa_index.html); they includeproliferation, migration and tube formation assays using HUVECs.

Initially the anti-angiogenic effect of selected standard agents isassessed as a positive control by adding them into the wells containingcultured endothelial cells. Such standard anti-angiogenic agents includethe fumigillin analog TNP-470 and paclitaxel that are available byrequest from NCI. The standard cell culture medium is usually used as anegative control. The experiments described below are repeated severaltimes as required to obtain statistically significant and reproducibleresults. Once the platform is calibrated and tested for the knownagents, the novel inhibitors are tested.

Cell Proliferation Assay

In these assays anti-angiogenic agents are tested for their ability toalter endothelial cell proliferation. A reduction in endothelial cellproliferation identifies an agent that inhibits angiogenesis. Theviability and metabolic activity of the cells is measured in thepresence of the anti-angiogenic peptides at different concentrations andvarious time steps. In one example, a cell proliferation reagent, MTT,is used in a substrate/assay that measures the metabolic activity ofviable cells. The assay is based on the reduction of the yellowtetrazolium salt, MTT, by viable, metabolically active cells to form theinsoluble purple formazan crystals, which are solubilized by theaddition of a detergent. MTT is a colorimetric, non-radioactive assaythat can be performed in a microplate. It is suitable for measuring cellproliferation, cell viability or cytotoxicity. The procedure involvesthree steps. First, the cells are cultured in a multi-well plate andthen incubated with the yellow MTT for approximately 2 to 4 hours.During this incubation period, viable cells convert, in theirmitochondria, the yellow MTT to the purple formazan crystals. The secondstep involves the solubilization of the crystals. A detergent solutionis added to lyse the cells and solubilize the colored crystals. Thefinal step of the assay involves quantifying changes in proliferation bymeasuring the changes in the color after lysing the cells. The samplesare read using an ELISA plate reader at a wavelength of 570 nm. Theamount of color produced is directly proportional to the number ofviable cells present in a particular well. Other proliferation assaysinclude WST-1, XTT, Trypan Blue, Alamar Blue and BrdU. In contrast tothe MTT assay, in the WST-1 assay the formazan crystals do not need tobe solubilized by the addition of a detergent; they are soluble to thecell medium.

In another example, cell proliferation is assayed by quantitatingbromodeoxyuridine (BrdU) incorporation into the newly synthesized DNA ofreplicating cells. The assay is a cellular immunoassay that uses a mousemonoclonal antibody directed against BrdU. The procedure involves foursteps. First, the cells are cultured in a microtiterplate andpulse-labeled with BrdU. Only proliferating cells incorporate BrdU intotheir DNA. The cells are then fixed in a denaturing solution. Thegenomic DNA is denatured, exposing the incorporated BrdU toimmunodetection. The BrdU label is located in the DNA with aperoxidase-conjugated anti-BrdU antibody. The antibody is quantitatedusing a peroxidase substrate. To test anti-proliferative effects of theselected peptides, the endothelial cells are incubated in the presenceof varying amounts of the peptides for different time intervals. Afterlabeling of the cells with BrdU the cell proliferation reagent WST-1 isadded, and the cells are reincubated. The formazan product is quantifiedat 450 nm with an absorbance reader. Subsequently, BrdU incorporation isdetermined using the colorimetric cell proliferation ELISA, BrdU. Theresults of this assay indicate the effects of the anti-angiogenicpeptides either on DNA synthesis (anti-proliferative) or the metabolicactivity (pro-apoptotic) of the cell. Kits for implementing thesetechniques are commercially available.

Preferably, an agent of the invention reduces cell proliferation by atleast about 5%, 10%, 20% or 25%. More preferably, cell proliferation isreduced by at least 50%, 75%, or even by 100%.

Cell Apoptosis and Cell Cycle Assay

Agents having anti-angiogenic activity can also be identified in assaythat measure the effect of a candidate agent on cell proliferation andsurvival using a mitogenic assay (incorporation of thymidine, or5-bromodeoxyuridine) that measures alterations in cell number (directcounts or indirect colorimetric evaluation). Agents that reduce cellproliferation, cell survival, or that increase cell death are identifiedas having anti-angiogenic activity. Cell death by apoptosis can bemeasured using a TUNEL assay or by analyzing the expression of apoptosismarkers, such as the caspases and annexin V (Fennell et al., J BiomolScreen, 11:296-302, 2006; Loo and Rillema, Methods Cell Biol, 57:251-64,1998; Otsuki et al., Prog Histochem Cytochem, 38:275-339, 2003).

A number of methods have been developed to study apoptosis in cellpopulations. Apoptosis is a form of cell death that is characterized bycleavage of the genomic DNA into discrete fragments prior to membranedisintegration. Because DNA cleavage is a hallmark for apoptosis, assaysthat measure prelytic DNA fragmentation are especially attractive forthe determination of apoptotic cell death. DNA fragments obtained fromcell populations are assayed on agarose gels to identify the presence ofabsence of “DNA ladders” or bands of 180 by multiples, which form therungs of the ladders, or by quantifying the presence of histonecomplexed DNA fragments by ELISA.

Other indicators of apoptosis include assaying for the presence caspasesthat are involved in the early stages of apoptosis. The appearance ofcaspases sets off a cascade of events that disable a multitude of cellfunctions. Caspase activation can be analyzed in vitro by utilizing anenzymatic assay. Activity of a specific caspase, for instance caspase 3,can be determined in cellular lysates by capturing of the caspase andmeasuring proteolytic cleavage of a suitable substrate that is sensitiveto the specific protease (Fennell et al., J Biomol Screen, 11:296-302,2006; Loo and Rillema, Methods Cell Biol, 57:251-64, 1998; Otsuki etal., Prog Histochem Cytochem, 38:275-339, 2003). Agents that increasecaspase activity or DNA fragmentation in endothelial cells areidentified as useful in the methods of the invention.

In addition to in vitro techniques, apoptosis can be measured using flowcytometry. One of the simplest methods is to use propidium iodide (PI)to stain the DNA and look for sub-diploid cells (Fennell et al., JBiomol Screen, 11:296-302, 2006; Loo and Rillema, Methods Cell Biol,57:251-64, 1998; Otsuki et al., Prog Histochem Cytochem, 38:275-339,2003).

The most commonly used dye for DNA content/cell cycle analysis ispropidium iodide (PI). PI intercalates into the major groove ofdouble-stranded DNA and produces a highly fluorescent adduct that can beexcited at 488 nm with a broad emission centered around 600 nm. Since PIcan also bind to double-stranded RNA, it is necessary to treat the cellswith RNase for optimal DNA resolution. Other flow cytometric-basedmethods include the TUNEL assay, which measures DNA strand breaks andAnnexin V binding, which detects relocation of membrane phosphatidylserine from the intracellular surface to the extracellular surface.

Cell Migration and Invasion Assay

Another anti-angiogenic activity is the ability to reduce endothelialcell migration towards an attractant that is present in a chemotacticgradient, such as a growth factor gradient. Endothelial cell motility ormigration can be assessed using the Boyden chamber technique (Auerbachet al., Cancer Metastasis Rev, 19:167-72, 2000; Auerbach et al., ClinChem, 49:32-40, 2003; Taraboletti and Giavazzi, Eur J Cancer, 40:881-9,2004). In one example, a Boyden chamber assay is used to testendothelial cell migration from one side of the chamber in the presenceof an activator. In brief, the lower compartment of the Boyden chamberis separated from the upper (containing the endothelial cells) by amatrix-coated polycarbonate filter with pores small enough to allow onlythe active passage of the cells (3-8 μm pore size). The matrix mayinclude, for example, extracellular matrix proteins, such as collagen,laminin and fibronectin. Activators include but are not limited togrowth factors, such as vascular endothelial growth factor andfibroblast growth factor-2 or conditioned medium (e.g. from tumor cellsor NIH-3T3 fibroblasts). Migration typically occurs rapidly typicallywithin 4-20 hours cells have migrated through the filter. The number ofmigrating cells is quantified using a cell-permeable fluorescent dye inthe presence or absence of an inhibitor; it can also be quantified byany means of cell counting. A fluorescence plate reader is used toquantify the migrating cells by measuring the amount of fluorescence anddirectly correlating it to cell number. A decrease in cell migrationidentifies a peptide that inhibits angiogenesis. Preferably, cellmigration or motility is reduced by at least about 5%, 10%, 20% or 25%.More preferably, cell migration or motility is reduced by at least about50%, 75%, or even by 100%.

In other embodiments, anti-angiogenic agents of the invention alter theinvasiveness of an endothelial cell, for example, by reducing theability of an endothelial cell to degrade an extracellular matrixcomponent. In one example, an anti-angiogenic inhibitor acts by reducingthe proteolytic activity of a matrix metalloproteinase. Methods forassaying protease activity are known in the art. Quantification of thematrix metalloproteinase activity can be accomplished using azymographic or gelatinase activity assay (Frederiks and Mook, JHistochem Cytochem. 52:711-22, 2004). Preferably, protease activity isreduced by at least about 5%, 10%, 20% or 25%. More preferably, proteaseactivity is reduced by at least about 50%, 75%, or even by 100%.

In another example, the invasive activity of an endothelial cell ismeasured using a Boyden chamber invasion assay or by measuringphagokinetic tracks. The invasion assay is essentially as describedabove for the Boyden motility assay, except that the filter is coatedwith a layer of a matrix several microns thick, usually Matrigel orother basement membrane extracts, which the cells must degrade beforemigrating through the filter (Auerbach et al., Cancer Metastasis Rev,19:167-72, 2000; Auerbach et al., Clin Chem, 49:32-40, 2003; Tarabolettiand Giavazzi, Eur J Cancer, 40:881-9, 2004). Compounds that reduceextracellular matrix degradation or endothelial cell invasiveness areidentified as useful in the methods of the invention.

Tube Formation Assay

Another method of identifying an agent having anti-angiogenic activityinvolves measuring the agent's ability to reduce or disrupt capillarytube formation. Various types of endothelial cells (e.g., HUVECs, HMVECs(human microvascular endothelial cells)) form tubes when cultured inwells uniformly coated with Matrigel, an extracellular matrix protein,or other substrates. Therefore the assay characterizes endothelial celldifferentiation. The endothelial cells are cultured in the presence orthe absence of a candidate agent. The agent may be added to the culturemedia or may be present or applied to the gel. Typically, the effect ontube formation is measured by incubating the cells for a period of time(e.g., one to four days) at 37° C. in 5% CO₂ atmosphere. Kits forimplementing these techniques are commercially available.

The output of the experiments are images of capillary networks formed. Acommon metric used for the morphological characteristics of a capillarynetwork is the angiogenic index. This index is calculated as the ratioof the total length of the connected tubes over the total monitoredsurface of the well. The change of the angiogenic index as a function ofthe concentration of the anti-angiogenic peptide will be the determinantfor the effectiveness of the tested novel angiogenesis inhibitors.

Aortic Ring Assay

The aortic ring assay integrates the advantages of both in vivo and invitro systems. It is a useful assay to test angiogenic factors orinhibitors in a controlled environment. More importantly, itrecapitulates all of the necessary steps involved in angiogenesis(Staton et al., Int J Exp Pathol, 85:233-48, 2004).

In this quantitative method of studying angiogenesis, ring segments ofaortas from various animals such as rats and mice are embedded in athree-dimensional matrix composed of fibrin or collagen, and cultured ina defined medium devoid of serum and growth factors. Microvessels sproutspontaneously from the surface of the aortic rings. This angiogenicprocess is mediated by endogenous growth factors produced from the aortaor can be assisted by applying exogenously specific concentrations ofgrowth factors. The embedded aortas are incubated for 10-12 days andafter the incubation period the newly formed vessels are quantified.Microvessels can be counted manually or quantified usingcomputer-assisted image analysis. Test agents can be added to theculture medium to assay for angiogenic or anti-angiogenic activity. Alsoaortas from animals with different genetic background (e.g., knockoutmice) can be used in order to assess specific mechanisms of the effectof the anti-angiogenic peptides on the neovessel formation process.

In Vivo Angiogenesis Assays

A recent review identified over 70 disease conditions that involveangiogenesis, about half of those characterized by abnormal or excessiveangiogenesis or lymphangiogenesis (Carmeliet, Nature, 438:932-6, 2005).Agents identified as having anti-angiogenic activity are optionallytested in in vivo assays using animal models that exhibit abnormal orexcessive angiogenesis or lymphangiogenesis.

Matrigel Plug Assay

In one in vivo approach, a candidate agent of the invention is testedfor anti-angiogenic activity by implanting a polymer matrixsubcutaneously in an animal and assaying the matrix for signs ofneovascularization. In one embodiment, a Matrigel plug or a similarsubstrate containing tumor cells and an anti-angiogenic factor is usedto study in vivo angiogenesis (Auerbach et al., Cancer Metastasis Rev,19:167-72, 2000; Staton et al., Int J Exp Pathol, 85:233-48, 2004).Matrigel is a liquid at 4° C., but forms a solid gel at 37° C. Acandidate agent is suspended together with an attractant, such as agrowth factor, in the gel. The Matrigel is then injected subcutaneouslywhere it forms a solid plug allowing for the prolonged local release ofpro- or anti-angiogenic agents present in the gel. The plug issubsequently removed and neovascularization is assessed by any standardmethods, including but not limited to, identifying the presence ofendothelial cells or endothelial cell tubules in the plug usingmicroscopy. In some embodiments, this approach is combined with animmuno-histological identification of endothelium specific proteins(e.g., CD-31/34 or integrins) on the newly formed vessels.

The Matrigel plug assay can be applied for testing the efficacy of thenovel anti-angiogenic peptides identified herein. In one example,Matrigel is mixed with heparin (usually 20 U/ml) and a vascularendothelial growth factor at about 50 ng/ml in the presence or absenceof a candidate peptide, which is supplied at a variety of concentrations(e.g., at the IC₅₀). A control animal receives the gel without theanti-angiogenic fragment. The Matrigel is injected into the micesubcutaneously and after one week the animals are sacrificed. TheMatrigel plugs are then removed and fixed with 4% paraformaldehyde. Theplugs are then embedded in paraffin, sectioned and stained withhematoxylin and eosin. The number of blood vessels as well as any otherangiogenic indexes are estimated.

Directed In Vivo Angiogenesis Assay (DIVAA)

Directed in vivo angiogenesis assay (DIVAA) is a reproducible andquantitative in vivo method of assaying angiogenesis. It involves thepreparation of silicon cylinders that are closed on one side filled withsome type of extracellular matrix (for example Matrigel) with or withoutpremixed angiogenic factors (Guedez et al., Am J Pathol, 162:1431-9,2003) to form an angioreactor. The angioreactors are then implantedsubcutaneously in mice. Vascular endothelial cells migrate into theextracellular matrix and form vessels in the angioreactor. As early asnine days post-implantation, there are enough cells present in theangioreactor to assay the effect of an angiogenic modulating factors. Acandidate agent may be included in the matrix together with theangiogenic factors. The design of the angioreactor provides astandardized platform for reproducible and quantifiable in vivoangiogenesis assays.

Advantageously, the angioreactor prevents assay errors due to absorptionof the basement membrane extract or the diffusion of the anti-angiogenicagent into the surrounding tissue; may be carried out using only afraction of the materials required in the plug assay described above;and up to four angioreactors may be implanted in a single animal (e.g.,mouse), providing more data for analysis. Vascularization response canbe measured by intravenous injection of fluorescein isothiocyanate(FITC)-dextran before the recovery of the angioreactor, followed byspectrofluorimetry. Alternatively, to obtain a quantitative assessmentof the angiogenic invasion, the content of the angioreactors, can beremoved and the endothelial cells stained using FITC-Lectin.Fluorescence of the FITC-Lectin solution can be quantitated by measuringthe fluorescence at 485 nm excitation and 510 nm emission using afluorescence plate reader e.g., Victor 3V (Perkin Elmer). The intensityof the signal is directly proportional to the number of endothelialcells that are present in the angioreactors. The technique allows doseresponse analysis and identification of effective doses ofangiogenesis-modulating factors in vivo.

Chorioallantoic Membrane Assay

The chorioallantoic membrane assay (CAM) is widely used as anangiogenesis assay Auerbach et al., Cancer Metastasis Rev 19:167-172,2000; Staton et al., Int J Exp Pathol 85: 233-248, 2004; D'Amato, In:Voest, E. E., and D'Amore, P. A. (eds). Tumor Angiogenesis andMicrocirculation, 2001, Marcel Dekker, New York-Basel). In oneembodiment, the chorioallantoic membrane of a 7-9 day old chick embryosis exposed by making a window in the egg shell. A candidate agent isprovided in a formulation that provides for its extended release (e.g.,in a slow-release polymer pellets, absorbed on a gelatin sponge, orair-dried onto a plastic disc). The candidate agent formulation isimplanted onto the chorioallantoic membrane through a window in theshell. The window is sealed and the egg is re-incubated. The lack ofmature immune system in the 7 day old chick embryos allows the study ofangiogenesis without any immunological interference. In the modifiedversion of the in ovo assay, the entire egg content is transferred to aplastic culture dish. After 3-6 days of incubation the testing agentsare applied and angiogenesis is monitored using various angiogenesisindexes.

In the case of testing the angiostatic peptides, polymer pellets can beloaded both with the growth factors and the anti-angiogenic fragmentsand be implanted in the chorioallantoic membrane. The modified versionof the assay allows the application of a candidate agent using differentstrategies to identify effective therapeutic regimens. For example, acandidate agent is applied in a single bolus at a particularconcentration; at different time points at lower concentrations; or indifferent formulations that provide for the extended release of anagent. This provides for the temporal control of candidate agent releaseand the delineation of temporal variations in drug administration on theangiostatic activity of the candidate agents.

Ocular Angiogenesis Models

The cornea is an avascular site and presumably any vessels penetratingfrom the limbus into the cornea stroma can be identified as newlyformed. In this assay a pocket is created in the cornea stroma of theanimal. An angiogenic response is usually initiated by implantation of aslow release pellet or polymer containing growth factors (Auerbach etal., Cancer Metastasis Rev, 19:167-72, 2000; Auerbach et al., Clin Chem,49:32-40, 2003; D'Amato, Tumor Angiogenesis and Microcirculation,103-110, 2001; Staton et al., Int J Exp Pathol, 85:233-48, 2004).

In order to test an angiogenesis inhibitor, the effect of a candidateagent on an angiogenic response in the cornea is assayed after theimplantation of a pellet comprising an angiogenic agent in combinationwith a candidate inhibitor in the cornea pockets. Also the efficacy ofan anti-angiogenic agent can be evaluated using the mouse model ofocular ischemic retinopathy to quantitatively assess antiangiogeniceffects on retinal neovascularization. In addition, a mouse model oflaser induced choroidal neovascularization can be used in order toquantitatively assess the anti-angiogenic effects of candidate agents onchoroidal neovascularization. The tested peptides can be administeredwith a bolus injection or any other scheduled administration.

Chamber Assays

Other methods for studying the effect of a candidate agent in vivo onchronic angiogenesis involve the use of an implanted transparentchamber. The chamber is implanted in an accessible site (e.g., therabbit ear, the dorsal skinfold and the cranial window chamber (Auerbachet al., Clin Chem, 49:32-40, 2003; Staton et al., Int J Exp Pathol,85:233-48, 2004). In each of these systems a piece of skin (the ear orskinfold chamber) or part of the skull (cranial chamber) is removed froman anesthetized animal. Tumor cells or a pellet containing anangiogenesis stimulus is then placed on the exposed surface and coveredby a glass. The animals are allowed to recover, and angiogenesis issubsequently monitored. The models allow for the continuous measurementof various angiogenesis as well as tissue parameters, such as pH orblood flow. Similarly to the corneal pocket assay, the angiostaticagents are administered orally, locally, or systemically using apredefined drug administration schedule. Agents that reduce angiogenesisin a chamber assay are identified as useful in the methods of theinvention.

Tumor Models

Many different in vivo models have been developed to test the activityof potential anti-angiogenic or anti-cancer treatments, specifically ontumor vasculature. Tumors are implanted and can be grown syngeneically;i.e., subcutaneously, orthotopically in a tissue of origin, or asxenografts in immunodeficient mice (Auerbach et al., Clin Chem,49:32-40, 2003; Staton et al., Int J Exp Pathol, 85:233-48, 2004). Anynumber of histological analyses may be used to examine the effect of acandidate agent on a blood vessel supplying the tumor. In oneembodiment, the blood vessel density of a newly formed vasculature inthe tumor is monitored; in another embodiment, the vascular architectureis monitored, for example, by counting the number of vascular branchesper vessel unit length. In another embodiment, blood flow through thevasculature is measured.

The tumor models provide a variety of different conditions that can beanalyzed to assay the efficacy of a candidate anti-angiogenic agent. Forexample, the effects of a candidate agent on the stability of a wellvascularized vs. a poorly vascularized tumor can be assayed; the effectof a candidate agent on tumors of different origin, for example prostateand breast cancer, renal cell carcinoma, and including those of vascularorigin such as the chemically induced hemangiosarcomas and Kaposi'ssarcomas, can be analyzed. The study of in vivo tumor models provide theclosest approximation of human tumor angiogenesis. Moreover, such modelsprovide the opportunity to study the pharmacokinetics of the candidatedrug as well as its efficacy simultaneously in a large scale model andunder different administration carriers and strategies.

Anti-Angiogenic Peptides and Analogs

The invention is not limited to conventional therapeutic peptides havinganti-angiogenic activity, but comprises a variety of modified peptideshaving properties that enhance their biodistribution, selectivity, orhalf-life. In particular, the invention provides peptides that aremodified in ways that enhance their ability to inhibit angiogenesis in acell, tissue, or organ in a subject in need thereof.

The invention provides methods for optimizing a transcription factor orprotein transduction domain amino acid sequence or nucleic acid sequenceby producing an alteration in the sequence. Such alterations may includecertain mutations, deletions, insertions, or post-translationalmodifications. The invention further includes analogs of anynaturally-occurring polypeptide of the invention. Analogs can differfrom a naturally-occurring polypeptide of the invention by amino acidsequence differences, by post-translational modifications, or by both.Analogs of the invention will generally exhibit at least 85%, morepreferably 90%, and most preferably 95% or even 99% identity with all orpart of a naturally-occurring amino, acid sequence of the invention. Thelength of sequence comparison is at least 5, 10, 15 or 20 amino acidresidues, preferably at least 25, 50, or 75 amino acid residues, andmore preferably more than 100 amino acid residues. Again, in anexemplary approach to determining the degree of identity, a BLASTprogram may be used, with a probability score between e⁻³ and e⁻¹⁰⁰indicating a closely related sequence. Modifications include in vivo andin vitro chemical derivatization of polypeptides, e.g., acetylation,carboxylation, phosphorylation, or glycosylation; such modifications mayoccur during polypeptide synthesis or processing or following treatmentwith isolated modifying enzymes. Analogs can also differ from thenaturally-occurring polypeptides of the invention by alterations inprimary sequence. These include genetic variants, both natural andinduced (for example, resulting from random mutagenesis by irradiationor exposure to ethanemethylsulfate or by site-specific mutagenesis asdescribed in Sambrook, Fritsch and Maniatis, Molecular Cloning: ALaboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra).Also included are cyclized peptides, molecules, and analogs whichcontain residues other than L-amino acids, e.g., D-amino acids ornon-naturally occurring or synthetic amino acids, e.g., β or γ aminoacids.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, for example,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine,phosphothreonine. “Amino acid analogs” refer to compounds that have thesame basic chemical structure as a naturally occurring amino acid, i.e.,a carbon that is bound to a hydrogen, a carboxyl group, an amino group,and an R group, for example, homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (for example, norleucine) or modified peptide backbones, butretain the same basic chemical structure as a naturally occurring aminoacid. “Amino acid mimetics” refers to chemical compounds that have astructure that is different from the general chemical structure of anamino acid, but that function in a manner similar to a naturallyoccurring amino acid. Amino acids and analogs are well known in the art.Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” apply to both amino acid and nucleicacid sequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or similar amino acid sequences and include degeneratesequences. For example, the codons GCA, GCC, GCG and GCU all encodealanine. Thus, at every amino acid position where an alanine isspecified, any of these codons can be used interchangeably inconstructing a corresponding nucleotide sequence. The resulting nucleicacid variants are conservatively modified variants, since they encodethe same protein (assuming that is the only alternation in thesequence). One skilled in the art recognizes that each codon in anucleic acid, except for AUG (sole codon for methionine) and UGG(tryptophan), can be modified conservatively to yield afunctionally-identical peptide or protein molecule. As to amino acidsequences, one skilled in the art will recognize that substitutions,deletions, or additions to a polypeptide or protein sequence whichalter, add or delete a single amino acid or a small number (typicallyless than about ten) of amino acids is a “conservatively modifiedvariant” where the alteration results in the substitution of an aminoacid with a chemically similar amino acid. Conservative substitutionsare well known in the art and include, for example, the changes ofalanine to serine; arginine to lysine; asparigine to glutamine orhistidine; aspartate to glutamate; cysteine to serine; glutamine toasparigine; glutamate to aspartate; glycine to proline; histidine toasparigine or glutamine; isoleucine to leucine or valine; leucine tovaline or isoleucine; lysine to arginine, glutamine, or glutamate;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucineor leucine. Other conservative and semi-conservative substitutions areknown in the art and can be employed in practice of the presentinvention.

The terms “protein”, “peptide” and “polypeptide” are used herein todescribe any chain of amino acids, regardless of length orpost-translational modification (for example, glycosylation orphosphorylation). Thus, the terms can be used interchangeably herein torefer to a polymer of amino acid residues. The terms also apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid.Thus, the term “polypeptide” includes full-length, naturally occurringproteins as well as recombinantly or synthetically produced polypeptidesthat correspond to a full-length naturally occurring protein or toparticular domains or portions of a naturally occurring protein. Theterm also encompasses mature proteins which have an added amino-terminalmethionine to facilitate expression in prokaryotic cells.

The polypeptides and peptides of the invention can be chemicallysynthesized or synthesized by recombinant DNA methods; or, they can bepurified from tissues in which they are naturally expressed, accordingto standard biochemical methods of purification. Also included in theinvention are “functional polypeptides,” which possess one or more ofthe biological functions or activities of a protein or polypeptide ofthe invention. These functions or activities include the ability toinhibit angiogenesis (e.g., by reducing endothelial cell proliferation,migration, survival, or tube formation). The functional polypeptides maycontain a primary amino acid sequence that has been modified from thatconsidered to be the standard sequence of a peptide described herein(e.g., SEQ ID Nos 1-156). Preferably these modifications areconservative amino acid substitutions, as described herein.

In addition to full-length polypeptides, the invention also includesfragments of any one of the polypeptides of the invention. As usedherein, the term “a fragment” means at least 5, 10, 13, or 15 aminoacids. In other embodiments a fragment is at least 20 contiguous aminoacids, at least 21, 22, 23, 24, or 25 contiguous amino acids, or atleast 30, 35, 40, or 50 contiguous amino acids, and in other embodimentsat least 60 to 80 or more contiguous amino acids. Fragments of theinvention can be generated by methods known to those skilled in the artor may result from normal protein processing (e.g., removal of aminoacids from the nascent polypeptide that are not required for biologicalactivity or removal of amino acids by alternative mRNA splicing oralternative protein processing events).

Non-protein transcription factor/protein transduction domain fusionanalogs have a chemical structure designed to mimic the fusion proteinsfunctional activity. Such analogs are administered according to methodsof the invention. Fusion protein analogs may exceed the physiologicalactivity of the original fusion polypeptide. Methods of analog designare well known in the art, and synthesis of analogs can be carried outaccording to such methods by modifying the chemical structures such thatthe resultant analogs increase the reprogramming or regenerativeactivity of a reference transcription factor/protein transduction domainfusion polypeptide. These chemical modifications include, but are notlimited to, substituting alternative R groups and varying the degree ofsaturation at specific carbon atoms of a reference fusion polypeptide.Preferably, the fusion protein analogs are relatively resistant to invivo degradation, resulting in a more prolonged therapeutic effect uponadministration. Assays for measuring functional activity include, butare not limited to, those described in the Examples below.

Peptide-Design Approaches

Iterative design approaches (DeFreest et al., J Pept Res, 63:409-19,2004) offer unique opportunities to optimize the structure and functionof the candidate anti-angiogenic peptides. During iterative design aninitial set of amino acids is substituted and the effect of theresulting agent on angiogenesis is assayed. The exploration of thestructure-function relationships, but most importantly the conservationof the biophysical and biochemical characteristics of the peptides,during the iterative design and synthesis, is expected to contribute tothe optimization of the anti-angiogenic activity. To determine whichresidues are essential to the bioactivity of the predicted peptide aseries of analogs is prepared and evaluated.

In order to assess the types of substitutions within the amino acidsequence of the candidate peptide one can initially use computationalmethods. The most straightforward method for deciphering the importanceof each amino acid is to investigate the conservation of these aminoacids at multiple orthologues (same locus in different organisms). Aminoacids that are conserved among different organisms are identified asfunctionally significant. From a biophysical point of view electrostaticinteractions and hydrophobic partitioning act in concert to promote theinteractions of the peptides with their receptors. In this sense, anypoint substitution should comply with the conservation of the net chargeand hydrophobicity of the agent (DeFreest et al., J Pept Res, 63:409-19,2004). Phage display technology can also be used for performing randomsubstitutions at expressed peptides of 20-25 amino acids length (Scottand Smith, Science, 249:386-90, 1990). In each of the cases theresultant peptide is tested for its effect on angiogenesis using any ofthe assays described herein.

Design optimization of the activity of the predicted peptides can alsobe performed by altering specific structural characteristics of theagents. For example, it has been shown (DeFreest et al., J Pept Res,63:409-19, 2004) that head-to-tail cyclization of the molecules confersan active dose range broader than the linear form of the molecules, andthe peptide stability and shelf life are not compromised. Thehead-to-tail conjunction can occur either by a disulfide bond or by apeptide bond formation. The use of a peptide bond may be advantageousfor purposes of shelf life, and elimination of dimers, trimers, andhigher-order aggregates formation that can sometimes develop whenpeptides are stored or used in conditions where the redox state cannotbe fully controlled. The cyclization approaches are discussed in thefollowing section.

Cyclization of Linear Peptides

Cyclization of peptides has been shown to be a useful approach todeveloping diagnostically and therapeutically useful peptidic andpeptidomimetic agents. Cyclization of peptides reduces theconformational freedom of these flexible, linear molecules, and oftenresults in higher receptor binding affinities by reducing unfavorableentropic effects. Because of the more constrained structural framework,these agents are more selective in their affinity to specific receptorcavities. By the same reasoning, structurally constrained cyclicpeptides confer greater stability against the action of proteolyticenzymes.

Methods for cyclization can be classified into the so called “backboneto backbone” cyclization by the formation of the amide bond between theN-terminal and the C-terminal amino acid residues, and cyclizationsinvolving the side chains of individual amino acids (Li and Roller, CurrTop Med Chem, 2:325-41, 2002). Although many novel approaches have beendeveloped to accomplish the head-to-tail cyclization of linear peptidesand peptidomimetics, the most commonly used method is still the solutionphase macro-cyclization using peptide coupling reagents. The results ofthe peptide cyclization are mainly influenced by the conformation of thelinear peptide precursors in solution. Synthesis design is affected bythe strategy of the ring disconnection, and the rational selection ofpeptide coupling reagents. A reasonable ring disconnection willsignificantly facilitate the peptide macro-cyclization reaction, while apoor selection of cyclization site may result in slow reaction speed andlow yield accompanied by various side reactions such as racemization,dimerization, and oligomerization.

Cyclization involving the side chains of individual amino acids includesthe formation of disulfide bridges between omega-thio amino acidresidues (cysteine, homocysteine), the formation of lactam bridgesbetween glutamic/aspartic acid and lysine residues, the formation oflactone or thiolactone bridges between amino acid residues containingcarboxyl, hydroxyl or mercapto functional groups, and the formation ofthio-ether or ether bridges between the amino acids containing hydroxylor mercapto functional groups.

Recombinant Polypeptide Expression

The invention provides therapeutic peptides that are most commonlygenerated by routine methods for peptide synthesis. Such methods areknown in the art and are described herein. If an alternative approach isdesired, the peptides are expressed recombinantly, either alone, or aspart of a larger fusion protein that includes an anti-angiogenic peptideoperably linked to a polypeptide that facilitates expression. Ifdesired, the peptide can subsequently be cleaved (e.g., enzymatically)from the fusion protein. Where the fusion protein does not interferewith the anti-angiogenic activity of the peptide such cleavage may notbe necessary or even desirable. When the therapeutic peptide or fusionprotein comprising the peptide contacts an endothelial cell, tissue, ororgan comprising such a cell it reduces angiogenesis. Recombinantpolypeptides of the invention are produced using virtually any methodknown to the skilled artisan. Typically, recombinant polypeptides areproduced by transformation of a suitable host cell with all or part of apolypeptide-encoding nucleic acid molecule or fragment thereof in asuitable expression vehicle.

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. A polypeptide of the invention may be produced in aprokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g.,Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammaliancells, e.g., NIH 3T3, HeLa, or preferably COS cells). Such cells areavailable from a wide range of sources (e.g., the American Type CultureCollection, Rockland, Md.; also, see, e.g., Ausubel et al., CurrentProtocol in Molecular Biology, New York: John Wiley and Sons, 1997). Themethod of transformation or transfection and the choice of expressionvehicle will depend on the host system selected. Transformation andtransfection methods are described, e.g., in Ausubel et al. (supra);expression vehicles may be chosen from those provided, e.g., in CloningVectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).

A variety of expression systems exist for the production of thepolypeptides of the invention. Expression vectors useful for producingsuch polypeptides include, without limitation, chromosomal, episomal,and virus-derived vectors, e.g., vectors derived from bacterialplasmids, from bacteriophage, from transposons, from yeast episomes,from insertion elements, from yeast chromosomal elements, from virusessuch as baculoviruses, papova viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof.

One particular bacterial expression system for polypeptide production isthe E. coli pET expression system (e.g., pET-28) (Novagen, Inc.,Madison, Wis). According to this expression system, DNA encoding apolypeptide is inserted into a pET vector in an orientation designed toallow expression. Since the gene encoding such a polypeptide is underthe control of the T7 regulatory signals, expression of the polypeptideis achieved by inducing the expression of T7 RNA polymerase in the hostcell. This is typically achieved using host strains that express T7 RNApolymerase in response to IPTG induction. Once produced, recombinantpolypeptide is then isolated according to standard methods known in theart, for example, those described herein.

Another bacterial expression system for polypeptide production is thepGEX expression system (Pharmacia). This system employs a GST genefusion system that is designed for high-level expression of genes orgene fragments as fusion proteins with rapid purification and recoveryof functional gene products. The protein of interest is fused to thecarboxyl terminus of the glutathione S-transferase protein fromSchistosoma japonicum and is readily purified from bacterial lysates byaffinity chromatography using Glutathione Sepharose 4B. Fusion proteinscan be recovered under mild conditions by elution with glutathione.Cleavage of the glutathione S-transferase domain from the fusion proteinis facilitated by the presence of recognition sites for site-specificproteases upstream of this domain. For example, proteins expressed inpGEX-2T plasmids may be cleaved with thrombin; those expressed inpGEX-3X may be cleaved with factor Xa.

Alternatively, recombinant polypeptides of the invention are expressedin Pichia pastoris, a methylotrophic yeast. Pichia is capable ofmetabolizing methanol as the sole carbon source. The first step in themetabolism of methanol is the oxidation of methanol to formaldehyde bythe enzyme, alcohol oxidase. Expression of this enzyme, which is codedfor by the AOX1 gene is induced by methanol. The AOX1 promoter can beused for inducible polypeptide expression or the GAP promoter forconstitutive expression of a gene of interest.

Once the recombinant polypeptide of the invention is expressed, it isisolated, for example, using affinity chromatography. In one example, anantibody (e.g., produced as described herein) raised against apolypeptide of the invention may be attached to a column and used toisolate the recombinant polypeptide. Lysis and fractionation ofpolypeptide-harboring cells prior to affinity chromatography may beperformed by standard methods (see, e.g., Ausubel et al., supra).Alternatively, the polypeptide is isolated using a sequence tag, such asa hexahistidine tag, that binds to nickel column.

Once isolated, the recombinant protein can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry and Molecular Biology,eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention,particularly short peptide fragments, can also be produced by chemicalsynthesis (e.g., by the methods described in Solid Phase PeptideSynthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Thesegeneral techniques of polypeptide expression and purification can alsobe used to produce and isolate useful peptide fragments or analogs(described herein).

Combinatorial Peptide Libraries

In addition to the synthetic solid state production of small peptides,the amino acid sequences of predicted fragments can be expressed andproduced recombinantly using a variety of genetically modified organismsfollowing insertion of the relevant DNA into their genome. One suchwidely used organism is Escherichia coli. Combinatorial biology dependson the ability to link peptides to their encoding DNA and create largelibraries of encoded peptides. The methods for generating DNA-encodedpeptide libraries can be divided into two groups. In vitro methods uselibraries in which the peptides are accessible to exogenous ligands orcells. These libraries can be used in direct in vitro binding selectionswith cell cultures to enrich for peptides that induce particularphenotypes. In contrast, in vivo methods use peptide libraries that areexpressed inside living cells. An interaction between a particularlibrary member and the target protein is detected by virtue of an effecton the host cell, such as a selective growth advantage, or changes to aphysical property of the host cell (Pelletier and Sidhu, Curr OpinBiotechnol, 12:340-7, 2001).

To optimize a set of peptides, such as those peptides identified herein,in vitro methods for creating and testing peptide libraries aresuitable. In one embodiment, oligonucleotide directed mutagenesis ofinitial sequence is used. In another embodiment, a phage is used todisplay libraries of peptides.

Oligonucleotide Directed Mutagenesis

Oligonucleotide directed mutagenesis can be used in order to modify asingle or multiple amino acids that compose the maternal sequence of thepredicted anti-angiogenic fragments (Ryu and Nam, Biotechnol Prog,16:2-16, 2000). Directed mutagenesis is based on the concept that anoligonucleotide encoding a desired mutation is annealed to one strand ofa DNA of interest and serves as a primer for initiation of DNAsynthesis. In this manner, the mutagenic oligonucleotide is incorporatedinto the newly synthesized strand. Mutagenic oligonucleotidesincorporate at least one base change but can be designed to generatemultiple substitutions, insertions or deletions.

Oligonucleotides can also encode a library of mutations by randomizingthe base composition at sites during chemical synthesis resulting indegenerate oligonucleotides. The ability to localize and specifymutations is greatly enhanced by the use of synthetic oligonucleotideshybridized to the DNA insert-containing plasmid vector. The generalformat for site-directed mutagenesis includes several steps. Plasmid DNAcontaining the template of interest (cDNA) is denatured to producesingle-stranded regions. A synthetic mutant oligonucleotide is annealedto the target strand. DNA polymerase is used to synthesize a newcomplementary strand, and finally DNA ligase is used to seal theresulting nick between the end of the new strand and theoligonucleotide. The resulting heteroduplex is propagated bytransformation in E. coli.

Phage-Displayed Peptide Library Screening

Phage display is one method for in vitro combinatorial biology. Themethod stems from the observation that peptides fused to certainbacteriophage coat proteins are displayed on the surfaces of phageparticles that also contain the cognate DNA (Landon et al., Curr DrugDiscov Technol, 1:113-32, 2004).

Phage display describes a selection technique in which a library ofvariants of an initial peptide (e.g., a peptide described herein), isexpressed on the outside of a phage virion, while the genetic materialencoding each variant resides on the inside. This creates a physicallinkage between each variant protein sequence and the DNA encoding it,which allows rapid partitioning based on binding affinity to a giventarget molecule by an in vitro selection process called panning. In itssimplest form, panning is carried out by incubating a library ofphage-displayed peptides with a plate containing a culture of cells,such as endothelial cells, washing away the unbound phage, and elutingthe specifically bound phage. The eluted phage is then amplified andtaken through additional binding/amplification cycles to enrich the poolin favor of specific phenotypes, such as suppression of proliferation,of the cells that are cultured. After 3-4 rounds, individual clones arecharacterized by DNA sequencing and ELISA.

Libraries of “fusion phages” are rapidly sorted to obtain clones withdesired properties and phages can be readily amplified by passagethrough a bacterial host. Phage display was first demonstrated with theEscherichia-coli-specific M13 bacteriophage and this remains the mostpopular platform. Several other E. coli phages have also been adaptedfor phage display and eukaryotic systems have also been developed.

Screening Assays

Polypeptides and fragments of the invention are useful as targets forthe identification of agents that modulate angiogenesis. In particular,the peptides identified herein (e.g., peptides listed in Table 1) aretypically polypeptide fragments that are hidden within hydrophobicregions of a larger polypeptide. While the entire polypeptide may bepro-angiogenic, the peptides of the invention are typicallyanti-angiogenic. As such, the activity of these peptides, when exposedto the cellular or extracellular milleau, may reduce the pro-angiogenicfunction of the larger polypeptide. Where this antagonistic function isundesirable, agents that bind and/or inhibit the biological activity ofthese peptides are sought. Once identified, such agents are used toenhance angiogenesis. In another approach, anti-angiogenic agents areidentified by screening for agents that bind to and enhance the activityof a peptide of the invention. Once identified, such agents are used toreduce angiogenesis.

Alternatively, or in addition, candidate agents may be identified thatspecifically bind to and inhibit a peptide of the invention. Theefficacy of such a candidate compound is dependent upon its ability tointeract with the peptide. Such an interaction can be readily assayedusing any number of standard binding techniques and functional assays(e.g., those described in Ausubel et al., supra). For example, acandidate compound may be tested in vitro for interaction and bindingwith a polypeptide of the invention and its ability to modulateangiogenesis may be assayed by any standard assays (e.g., thosedescribed herein).

Potential antagonists include organic molecules, peptides, peptidemimetics, polypeptides, nucleic acid ligands, aptamers, and antibodiesthat bind to a peptide of the invention and thereby inhibit orextinguish its activity. Potential antagonists also include smallmolecules that bind to and occupy the binding site of the polypeptidethereby preventing binding to cellular binding molecules, such thatnormal biological activity is prevented.

In one particular example, a candidate compound that binds to apathogenicity polypeptide may be identified using a chromatography-basedtechnique. For example, a recombinant polypeptide of the invention maybe purified by standard techniques from cells engineered to express thepolypeptide, or may be chemically synthesized, once purified the peptideis immobilized on a column. A solution of candidate compounds is thenpassed through the column, and a compound specific for the peptide isidentified on the basis of its ability to bind to the peptide and beimmobilized on the column. To isolate the compound, the column is washedto remove non-specifically bound molecules, and the compound of interestis then released from the column and collected. Compounds isolated bythis method (or any other appropriate method) may, if desired, befurther purified (e.g., by high performance liquid chromatography). Inaddition, these candidate compounds may be tested for their ability tomodulate angiogenesis (e.g., as described herein). Compounds isolated bythis approach may also be used, for example, as therapeutics to treat orprevent the onset of a disease or disorder characterized by excess orundesirable angiogenesis. Compounds that are identified as binding topeptides with an affinity constant less than or equal to 1 nM, 5 nM, 10nM, 100 nM, 1 mM or 10 mM are considered particularly useful in theinvention.

Methods of the invention are useful for the high-throughput low-costscreening of polypeptides, biologically active fragments or analogsthereof that can be used to modulate angiogenesis. One skilled in theart appreciates that the effects of a candidate peptide on a cell (e.g.,an endothelial cell) are typically compared to a corresponding controlcell not contacted with the candidate peptide. Thus, the screeningmethods include comparing the expression profile, phenotype, orbiological activity of a cell modulated by a candidate peptide to areference value of an untreated control cell.

In one example, candidate peptides are added at varying concentrationsto the culture medium of an endothelial cell. The survival, tubeformation, apoptosis, proliferation, migration of the cell are assayedas indicators of angiogenesis. Peptides that reduce the survival, tubeformation, proliferation, or migration of an endothelial cell areidentified as useful anti-angiogenic agents. Alternatively, peptidesthat enhance the survival, tube formation, proliferation, or migrationof an endothelial cell are identified as useful angiogenic agents. Inanother embodiment, the expression of a nucleic acid molecule orpolypeptide characteristic of the vasculature is monitored. Typical cellsurface markers include the fibronectin extra-domain B, large tenascin-Cisoforms, various integrins, VEGF receptors, prostate specific membraneantigen, endoglin and CD44 isoforms and tumor endothelium marker (TEM).Peptides or other agents that alter the expression of such markers areidentified as useful modulators of angiogenesis. An agent that reducesthe expression of a characteristic polypeptide expressed in thevasculature is considered useful in the invention; such an agent may beused, for example, as a therapeutic to prevent, delay, ameliorate,stabilize, or treat an injury, disease or disorder characterized by anundesirable increase in neovascularization. In other embodiments, agentsthat increase the expression or activity of a marker characteristicallyexpressed in an endothelial cell are used to prevent, delay, ameliorate,stabilize, or treat an injury, disease or disorder characterized by areduction in angiogenesis. Agents identified according to the methodsdescribed herein maybe administered to a patient in need of angiogenesismodulation. Where such agents are peptides, such as those describedherein, one skilled in the art appreciates that the invention furtherprovides nucleic acid sequences encoding these peptides (e.g., thoselisted in Table 1).

Test Compounds and Extracts

In general, peptides are identified from large libraries of naturalproduct or synthetic (or semi-synthetic) extracts or chemical librariesor from polypeptide or nucleic acid libraries, according to methodsknown in the art. Such candidate polypeptides or the nucleic acidmolecules encoding them may be modified to enhance biodistribution,protease resistance, or specificity. The modified peptides are thenscreened for a desired activity (e.g., angiogenesis modulatingactivity). Those skilled in the field of drug discovery and developmentwill understand that the precise source of test extracts or compounds isnot critical to the screening procedure(s) of the invention. Agents usedin screens may include known compounds (for example, known polypeptidetherapeutics used for other diseases or disorders). Alternatively,virtually any number of unknown chemical extracts or compounds can bescreened using the methods described herein. Examples of such extractsor compounds include, but are not limited to, plant-, fungal-,prokaryotic- or animal-based extracts, fermentation broths, andsynthetic compounds, as well as the modification of existingpolypeptides.

Libraries of natural polypeptides in the form of bacterial, fungal,plant, and animal extracts are commercially available from a number ofsources, including Biotics (Sussex, UK), Xenova (Slough, UK), HarborBranch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A.(Cambridge, Mass.). Such polypeptides can be modified to include aprotein transduction domain using methods known in the art and describedherein. In addition, natural and synthetically produced libraries areproduced, if desired, according to methods known in the art, e.g., bystandard extraction and fractionation methods. Examples of methods forthe synthesis of molecular libraries can be found in the art, forexample in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993;Erb et al., Proc. Natl. Acad. Sci. USA 91:11422, 1994; Zuckermann etal., J. Med. Chem. 37:2678, 1994; Cho et al., Science 261:1303, 1993;Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell etal., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al., J.Med. Chem. 37:1233, 1994. Furthermore, if desired, any library orcompound is readily modified using standard chemical, physical, orbiochemical methods.

Numerous methods are also available for generating random or directedsynthesis (e.g., semi-synthesis or total synthesis) of any number ofpolypeptides, chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic compound libraries are commercially available from BrandonAssociates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.).Alternatively, chemical compounds to be used as candidate compounds canbe synthesized from readily available starting materials using standardsynthetic techniques and methodologies known to those of ordinary skillin the art. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds identified by the methods described herein are known in theart and include, for example, those such as described in R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nded., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof.

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84,1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S.Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids(Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage(Scott and Smith, Science 249:386-390, 1990; Devlin, Science249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382,1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their activity should be employed wheneverpossible.

When a crude extract is found to have angiogenesis modulating activityfurther fractionation of the positive lead extract is necessary toisolate molecular constituents responsible for the observed effect.Thus, the goal of the extraction, fractionation, and purificationprocess is the careful characterization and identification of a chemicalentity within the crude extract that alters angiogenesis (increases ordecreases). Methods of fractionation and purification of suchheterogeneous extracts are known in the art. If desired, compounds shownto be useful as therapeutics are chemically modified according tomethods known in the art.

Therapeutic Methods

Therapeutic polypeptides, peptides, or analogs or fragments thereof, aswell as the nucleic acid molecules encoding such molecules are usefulfor preventing or ameliorating a disease or injury associated with anundesirable increase or decrease in angiogenesis. Diseases and disorderscharacterized by excess angiogenesis may be treated using the methodsand compositions of the invention. Such diseases and disorders include,but are not limited to, neoplasia, hematologic malignancies, rheumatoidarthritis, diabetic retinopathy, age-related macular degeneration,atherosclerosis, and pathologic obesity. In one embodiment, a peptide ofthe invention is delivered to one or more endothelial cells at a site ofangiogenesis-associated disease or injury.

In other embodiments, a nucleic acid molecule encoding a peptide of theinvention (e.g., a peptide listed in Table 1) is administered to a cell,tissue, or organ in need of a reduction in angiogenesis. If desired, thepeptide is expressed as a fusion with a longer polypeptide. The peptidemay then be cleaved from the polypeptide to achieve its desiredtherapeutic effect. Such cleavage is not required where the fusionprotein does not interfere with the peptide's biological activity.

Transducing viral (e.g., retroviral, adenoviral, and adeno-associatedviral) vectors can be used for somatic cell gene therapy, especiallybecause of their high efficiency of infection and stable integration andexpression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430,1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer etal., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A.94:10319, 1997). For example, a full length gene sialidase gene, or aportion thereof, can be cloned into a retroviral vector and expressioncan be driven from its endogenous promoter, from the retroviral longterminal repeat, or from a promoter specific for a target cell type ofinterest (e.g. endothelial cell). Other viral vectors that can be usedinclude, for example, a vaccinia virus, a bovine papilloma virus, or aherpes virus, such as Epstein-Barr Virus (also see, for example, thevectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988;Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990;Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic AcidResearch and Molecular Biology 36:311-322, 1987; Anderson, Science226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al.,Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science259:988-990, 1993; and Johnson, Chest 107:77 S-83S, 1995). Retroviralvectors are particularly well developed and have been used in clinicalsettings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson etal., U.S. Pat. No. 5,399,346). Most preferably, a viral vector is usedto administer the gene of interest systemically or to a cell at the siteof neovascularization.

Non-viral approaches can also be employed for the introduction oftherapeutic to a cell of a patient having an angiogenesis relateddisease. For example, a nucleic acid molecule can be introduced into acell by administering the nucleic acid in the presence of lipofectin(Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono etal., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci.298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983),asialoorosomucoid-polylysine conjugation (Wu et al., Journal ofBiological Chemistry 263:14621, 1988; Wu et al., Journal of BiologicalChemistry 264:16985, 1989), or by micro-injection under surgicalconditions (Wolff et al., Science 247:1465, 1990). Preferably thenucleic acids are administered in combination with a liposome andprotamine.

Gene transfer can also be achieved using non-viral means involvingtransfection in vitro. Such methods include the use of calciumphosphate, DEAE dextran, electroporation, and protoplast fusion.Liposomes can also be potentially beneficial for delivery of DNA into acell. Transplantation of normal genes into the affected tissues of apatient can also be accomplished by transferring a normal nucleic acidinto a cultivatable cell type ex vivo (e.g., an autologous orheterologous primary cell or progeny thereof), after which the cell (orits descendants) are injected into a targeted tissue at the site ofdisease or injury.

cDNA expression for use in such methods can be directed from anysuitable promoter (e.g., the human cytomegalovirus (CMV), simian virus40 (SV40), or metallothionein promoters), and regulated by anyappropriate mammalian regulatory element. For example, if desired,enhancers known to preferentially direct gene expression in specificcell types, such as an intestinal epithelial cell, can be used to directthe expression of a nucleic acid. The enhancers used can include,without limitation, those that are characterized as tissue- orcell-specific enhancers. Alternatively, if a genomic clone is used as atherapeutic construct, regulation can be mediated by the cognateregulatory sequences or, if desired, by regulatory sequences derivedfrom a heterologous source, including any of the promoters or regulatoryelements described above.

Another therapeutic approach included in the invention involvesadministration of a recombinant therapeutic, such as a sialidasepolypeptide, biologically active fragment, or variant thereof, eitherdirectly to the site of a potential or actual disease-affected tissue(for example, by administration to the intestine) or systemically (forexample, by any conventional recombinant protein administrationtechnique). The dosage of the administered protein depends on a numberof factors, including the size and health of the individual patient. Forany particular subject, the specific dosage regimes should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions. Generally, between 0.1 mg and 100 mg, is administered perday to an adult in any pharmaceutically acceptable formulation.

Pharmaceutical Therapeutics

The invention provides a simple means for identifying compositions(including nucleic acids, peptides, small molecule inhibitors, andmimetics) capable of acting as therapeutics for the treatment of adisease associated with altered levels of angiogenesis. Accordingly, achemical entity discovered to have medicinal value using the methodsdescribed herein is useful as a drug or as information for structuralmodification of existing compounds, e.g., by rational drug design. Suchmethods are useful for screening compounds having an effect on a varietyof conditions characterized by undesired angiogenesis.

For therapeutic uses, the compositions or agents identified using themethods disclosed herein may be administered systemically, for example,formulated in a pharmaceutically-acceptable buffer such as physiologicalsaline. Preferable routes of administration include, for example,subcutaneous, intravenous, interperitoneally, intramuscular, orintradermal injections that provide continuous, sustained levels of thedrug in the patient. Treatment of human patients or other animals willbe carried out using a therapeutically effective amount of a therapeuticagent described herein in a physiologically-acceptable carrier. Suitablecarriers and their formulation are described, for example, inRemington's Pharmaceutical Sciences by E. W. Martin. The amount of thetherapeutic agent to be administered varies depending upon the manner ofadministration, the age and body weight of the patient, and with theclinical symptoms of the disease or disorder. Generally, amounts will bein the range of those used for other agents used in the treatment ofother diseases associated with alterations in angiogenesis, although incertain instances lower amounts will be needed because of the increasedspecificity of the compound. A compound is administered at a dosage thatcontrols the clinical or physiological symptoms associated withangiogenesis as determined by a diagnostic method known to one skilledin the art.

It would be advantageous to administer therapeutic peptides in aformulation that would slow their elimination from the circulationthrough renal filtration, enzymatic degradation, uptake by thereticulo-endothelial system (RES), and accumulation in non-targetedorgans and tissues. In addition, methods for administering agents thatlimits their widespread distribution in non-targeted organs and tissuesallows lower concentrations of the agent to be administered reducingadverse side-effects and providing economic benefits. A variety ofmethods are available to slow the elimination of agents of theinvention. In one embodiment, an implantable device is used to providefor the controlled release of an agent described herein. Such devicesare known in the art and include, but are not limited to, polymeric gelsand micro-fabricated chips. Some of these devices are already used inthe clinic or are being tested in clinical trials (Moses et al., CancerCell, 4:337-41, 2003). Various delivery methods for anti-angiogenicagents are tissue specific, e.g., intraocular and periocular injectionor gene transfer in the eye (Akiyama et al., J Cell Physiol, 2006;Saishin et al., Hum Gene Ther, 16:473-8, 2005). Numerous reviews on thesubject of anti-angiogenic drug delivery are available.

Enhanced Permeability and Retention Effect

For the treatment of neoplasia or sites of neovascularization, the“enhanced permeability and retention effect” (EPR) constitutes a naturalmechanism through which high molecular weight (40 kDa or higher)macromolecules with long circulation half-lives, including peptides andproteins conjugated with water-soluble polymers, accumulate (Shukla andKrag, Expert Opin Biol Ther, 6:39-54, 2006; Torchilin and Lukyanov, DrugDiscov Today, 8:259-66, 2003). This effect occurs because of certaincharacteristics of those tissues. The first is that tumor or newlyformed vasculature, unlike the vasculature of healthy tissues, ispermeable to macromolecules with a MW up to 50 kDa or even higher. Thisallows macromolecules to enter into the interstitial space. Anothercharacteristic is that in the case of many tumors the lymphatic system,which is responsible for the drainage of macromolecules from normaltissues, is impaired. Because of this, macromolecules that have entereda neo-vascularized tissue are retained there for a prolonged time. Toenhance the retention of a low MW peptide described herein, the peptidemay be conjugated to a suitable polymer or delivered using amicro-reservoir system.

Peptide and Protein Polymer Conjugation

Mechanisms that increase the MW of a peptide, such as conjugation withpolymer chains or concentration of the drug in micro-reservoir systemstend to increase the retention time of the peptide in the tissue(Duncan, Nat Rev Drug Discov, 2:347-60, 2003). Moreover, renalfiltration and excretion are mainly responsible for the rapid clearancefrom the systemic circulation of proteins with molecular weights (MW) of40 kDa or lower. Rapid clearance and increased retention of a peptide ofinterest can be achieved by conjugating the peptides with water-solublepolymers. Preferably, the peptide-polymer conjugate has a molecularweight of at least about 30 kDA, 35 kDa, 40 kDa, 50 kDa, 75 kDa, or 100kDa. Additional benefits of peptide and protein-polymer conjugationinclude increased resistance to enzymatic degradation and reducedimmunogenicity.

Even endogenous proteins can be susceptible to protease degradation inthe bloodstream and interstitial space or induce an immune response.Enzymatic degradation and an immune response against a protein result inits rapid elimination from the systemic circulation. In addition, thedevelopment of an immune response is potentially dangerous because ofthe possibility of allergic reactions and anaphylactic shock uponrepetitive administrations. The mechanism of protein protection bypolymer attachment is similar in both cases. Polymer molecules attachedto the protein create steric hindrances, which interfere with binding tothe active sites of proteases, and antigen-processing cells. Examples ofpeptide/protein-polymer conjugation include conjugates withpoly(ethylene glycol) and conjugates with poly(styrene-co-maleic acidanhydride).

Conjugates with Poly(Ethylene Glycol)

Several polymers have been used for protein stabilization with varyingdegrees of success. Poly(ethylene glycol) (PEG) is one widely usedpolymer for the modification of proteins with therapeutic potential(Thanou and Duncan, Curr Opin Investig Drugs, 4:701-9, 2003; Vicent andDuncan, Trends Biotechnol, 24:39-47, 2006). This polymer is inexpensive,has low toxicity and has been approved for internal applications by drugregulatory agencies. PEG is commercially available in a variety ofmolecular weights and in chemically activated, ready-for-use forms forcovalent attachment to proteins.

Conjugates with Poly(Styrene-Co-Maleic Acid Anhydride)

In some cases, the circulation time of drugs can be increased byconjugating with polymers that are not large enough to prevent renalclearance themselves, but which can attach themselves, with theirconjugated drug, to natural long-circulating blood plasma components,such as serum albumin or lipoproteins (Thanou and Duncan, Curr OpinInvestig Drugs, 4:701-9, 2003; Vicent and Duncan, Trends Biotechnol,24:39-47, 2006).

Because of the small size and low molecular weight of the identifiedanti-angiogenic peptides and the high probability that the conjugatedpolymers, which are orders of magnitude larger than the peptides, maysterically hinder the activity of the fragments the method of proteinconjugation may not be the most efficient method for increasing theretention of the agent in the neo-vascular site. A more attractivescenario is the administration of the peptide in a micro-reservoirdelivery system.

Formulation of Pharmaceutical Compositions

The administration of a compound for the treatment of treatment of adisease or disorder associated with altered levels of angiogenesis maybe by any suitable means that results in a concentration of thetherapeutic that, combined with other components, is effective inameliorating, reducing, or stabilizing a disease or disorder associatedwith altered levels of angiogenesis (e.g., an amount sufficient toreduce neovascularization). The compound may be contained in anyappropriate amount in any suitable carrier substance, and is generallypresent in an amount of 1-95% by weight of the total weight of thecomposition. The composition may be provided in a dosage form that issuitable for parenteral (e.g., subcutaneously, intravenously,intramuscularly, or intraperitoneally) administration route. Thepharmaceutical compositions may be formulated according to conventionalpharmaceutical practice (see, e.g., Remington: The Science and Practiceof Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams &Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the active compound substantially immediately uponadministration or at any predetermined time or time period afteradministration. The latter types of compositions are generally known ascontrolled release formulations, which include (i) formulations thatcreate a substantially constant concentration of the drug within thebody over an extended period of time; (ii) formulations that after apredetermined lag time create a substantially constant concentration ofthe drug within the body over an extended period of time; (iii)formulations that sustain action during a predetermined time period bymaintaining a relatively, constant, effective level in the body withconcomitant minimization of undesirable side effects associated withfluctuations in the plasma level of the active substance (sawtoothkinetic pattern); (iv) formulations that localize action by, e.g.,spatial placement of a controlled release composition adjacent to or inthe central nervous system or cerebrospinal fluid; (v) formulations thatallow for convenient dosing, such that doses are administered, forexample, once every one or two weeks; and (vi) formulations that allowfor convenient dosing for metronomic therapy that would require takingsmall doses of the drug several times a week; (vii) formulations thattarget a disease or disorder associated with altered levels ofangiogenesis by using carriers or chemical derivatives to deliver thetherapeutic agent to a particular cell type (e.g., endothelial cell)whose function is perturbed in a disease or disorder associated withaltered levels of angiogenesis.

For some applications, controlled release formulations obviate the needfor frequent dosing during the day to sustain the plasma level at atherapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the therapeutic is formulatedwith appropriate excipients into a pharmaceutical composition that, uponadministration, releases the therapeutic in a controlled manner.Examples include single or multiple unit tablet or capsule compositions,oil solutions, suspensions, emulsions, microcapsules, microspheres,molecular complexes, nanoparticles, patches, and liposomes.

Micro-Reservoir Delivery Systems

For some applications, micro-reservoir or micro-particulate carriers areused to deliver a peptide of the invention. Such systems include, butare not limited to, liposomes, micelles, polymer micro-particles, andcell ghosts. The use of such carriers results in a much higher ratio ofactive agent over carrier compared with direct molecular conjugates.They also provide a higher degree of protection against enzymaticdegradation and other destructive factors upon parenteral administrationbecause the carrier wall completely isolates drug molecules from theenvironment. An additional advantage of these carriers is that a singlecarrier can deliver multiple drug species so that they can be used incombination therapies. All micro-particulates are too large to be lostby renal filtration (Thanou and Duncan, Curr Opin Investig Drugs,4:701-9, 2003) Exemplary micro-particulate delivery systems include, butare not limited to, liposomes and micelles.

Liposomes

Among particulate drug carriers, liposomes are the most extensivelystudied and possess suitable characteristics for peptide and proteinencapsulation. Liposomes are vesicles formed by concentric sphericalphospholipid bilayers encapsulating an aqueous space (Moses et al.,Cancer Cell, 4:337-41, 2003). These particles are biocompatible,biologically inert and cause little toxic or antigenic reactions. Theirinner aqueous compartment can be used for encapsulation of peptides andproteins. Many techniques for liposome preparation require onlymanipulations that are compatible with peptide and protein integrity(Allen and Cullis, Science, 303:1818-22, 2004). As with othermicro-particulate delivery systems, cells of the RES rapidly eliminateconventional liposomes.

In one embodiment, surface-modified long-circulating liposomes graftedwith a flexible hydrophilic polymer, such as PEG, are used. Thisapproach prevents plasma protein adsorption to the liposome surface andthe subsequent recognition and uptake of liposomes by the RES.Liposomes, in common with protein conjugated macromolecules, canaccumulate in tumors of various origins via the EPR effect. Currently,liposomal forms of at least two conventional anticancer drugs,daunorubicin and doxorubicin, are used in the clinic (Torchilin andLukyanov, Drug Discov Today, 8:259-66, 2003).

Micelles

In another approach, micelles or polymeric micelles, including thoseprepared from amphiphilic PEG-phospholipid conjugates, may be used todeliver an agent of the invention. Such formulations are of specialinterest because of their stability (Torchilin and Lukyanov, Drug DiscovToday, 8:259-66, 2003). These particles are smaller than liposomes andlack the internal aqueous space. To load micelles, peptides can beattached to the surface of these particles or incorporated into them viaa chemically attached hydrophobic anchor. An example of a biodegradablemicelle developed for delivery of pharmacological agents are thepoly{[(cholesteryl oxocarbonylamido ethyl)methyl bis(ethylene) ammoniumiodide]ethyl phosphate} (PCEP) micelles (Wen, Mao et al., J Pharm Sci.93:2142-57, 2004). Carrying a positive charge in its backbone and alipophilic cholesterol structure in the side chain, PCEP self-assemblesinto micelles in aqueous buffer at room temperature with an average sizeof 60-100 nm. PCEP is an excellent platform for deliveringant-angiogenic agents as by itself shows lower cytotoxicity forendothelial cells than for poly-L-lysine and Lipofectamine.

Nanoparticles

An increasing number of agents are delivered with colloidalnanoparticles. Such formulations overcome non-cellular and cellularbased mechanisms of resistance and increase the selectivity of agents totarget cells while reducing their toxicity in normal tissues.Nanoparticles are typically submicron (<1 μm) colloidal systems. In someembodiments, nanoparticles are made of polymers (biodegradable or not).According to the process used for the preparation of the nanoparticles,nanospheres or nanocapsules can be obtained. Unlike nanospheres (matrixsystems in which the drug is dispersed throughout the particles),nanocapsules are vesicular systems in which an agent is confined to anaqueous or oily cavity surrounded by a single polymeric membrane.Nanocapsules are one form of ‘reservoir’ system.

In some embodiments, nanoparticles are generated using hydrophilicpolymers, (poly(ethylene glycol) (PEG), poloxamines, poloxamers,polysaccharides) to efficiently coat a nanoparticle surface. Thesecoatings provide a dynamic ‘cloud’ of hydrophilic and neutral chains atthe particle surface that repels plasma proteins. Hydrophilic polymersare introduced at the surface in two ways, either by adsorption ofsurfactants or by use of block or branched copolymers.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in the form of a solution, a suspension, an emulsion,an infusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active therapeutic(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active therapeutic(s) may be incorporated intomicrospheres, microcapsules, nanoparticles, liposomes, or the like forcontrolled release. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in the form suitable for sterile injection. To preparesuch a composition, the suitable active angiogenic modulatingtherapeutic(s) are dissolved or suspended in a parenterally acceptableliquid vehicle. Among acceptable vehicles and solvents that may beemployed are water, water adjusted to a suitable pH by addition of anappropriate amount of hydrochloric acid, sodium hydroxide or a suitablebuffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloridesolution and dextrose solution. The aqueous formulation may also containone or more preservatives (e.g., methyl, ethyl or n-propylp-hydroxybenzoate). In cases where one of the compounds is onlysparingly or slightly soluble in water, a dissolution enhancing orsolubilizing agent can be added, or the solvent may include 10-60% w/wof propylene glycol or the like.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the active drugmay be incorporated in biocompatible carriers, liposomes, nanoparticles,implants, or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutam-nine) and, poly(lactic acid). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Solid Dosage Forms For Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. Such formulations are known to the skilled artisan.Excipients may be, for example, inert diluents or fillers (e.g.,sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starchesincluding potato starch, calcium carbonate, sodium chloride, lactose,calcium phosphate, calcium sulfate, or sodium phosphate); granulatingand disintegrating agents (e.g., cellulose derivatives includingmicrocrystalline cellulose, starches including potato starch,croscarmellose sodium, alginates, or alginic acid); binding agents(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodiumalginate, gelatin, starch, pregelatinized starch, microcrystallinecellulose, magnesium aluminum silicate, carboxymethylcellulose sodium,methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents,glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate,stearic acid, silicas, hydrogenated vegetable oils, or talc). Otherpharmaceutically acceptable excipients can be colorants, flavoringagents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drug ina predetermined pattern (e.g., in order to achieve a controlled releaseformulation) or it may be adapted not to release the active drug untilafter passage of the stomach (enteric coating). The coating may be asugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methyl hydroxyethylcellulose,hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers,polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating(e.g., based on methacrylic acid copolymer, cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate succinate, polyvinyl acetate phthalate, shellac, and/orethylcellulose). Furthermore, a time delay material such as, e.g.,glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active angiogenic modulatingtherapeutic). The coating may be applied on the solid dosage form in asimilar manner as that described in Encyclopedia of PharmaceuticalTechnology, supra.

At least two active angiogenic modulating therapeutics may be mixedtogether in the tablet, or may be partitioned. In one example, the firstactive in angiogenic modulating therapeutic is contained on the insideof the tablet, and the second active angiogenic modulating therapeuticis on the outside, such that a substantial portion of the secondangiogenic modulating therapeutic is released prior to the release ofthe first angiogenic modulating therapeutic.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders and granulates may be prepared using the ingredients mentionedabove under tablets and capsules in a conventional manner using, e.g., amixer, a fluid bed apparatus or a spray drying equipment.

Controlled Release Oral Dosage Forms

Controlled release compositions for oral use are constructed to releasethe active angiogenic modulating therapeutic by controlling thedissolution and/or the diffusion of the active substance. Dissolution ordiffusion controlled release can be achieved by appropriate coating of atablet, capsule, pellet, or granulate formulation of compounds, or byincorporating the compound into an appropriate matrix. A controlledrelease coating may include one or more of the coating substancesmentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax,carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryldistearate, glycerol palmitostearate, ethylcellulose, acrylic resins,dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride,polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3butylene glycol, ethylene glycol methacrylate, and/or polyethyleneglycols. In a controlled release matrix formulation, the matrix materialmay also include, e.g., hydrated methylcellulose, carnauba wax andstearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more therapeuticcompounds may also be in the form of a buoyant tablet or capsule (i.e.,a tablet or capsule that, upon oral administration, floats on top of thegastric content for a certain period of time). A buoyant tabletformulation of the compound(s) can be prepared by granulating a mixtureof the compound(s) with excipients and 20-75% w/w of hydrocolloids, suchas hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Polymeric Controlled-Release Implants

In another embodiment, an agent of the invention is delivered byimplanting a biodegradable polymeric controlled-release device thatstores the pharmaceutical agent and allows its delivery via diffusioninto the surrounding tissue. Controlled release devices include Norplantand Gliadel, which are used clinically for the prevention of pregnancyand for brain tumor therapy, respectively. Local delivery of pro- oranti-angiogenic factors can be accomplished by encapsulating the agentwithin a biocompatible polymer matrix. The controlled-release polymersystem is then implanted at the desired tissue site, where it releasesthe soluble factor directly into the interstitial space of the tissue.The diffusible agent can influence the survival or function of damagedcells within the local tissue, or provide a signal that elicits cellproliferation and migration or apoptosis and suppression of migrationwithin the tissue region.

Controlled release implants are typically composed of inert,biocompatible polymers, such as poly(ethylene-co-vinyl acetate) (EVAc),or biodegradable polymers, such as poly(lactide-co-glycolide) (PLGA)(Torchilin and Lukyanov, Drug Discov Today, 8:259-66, 2003). EVAc-matrixsystems have been used to release protein hormones, growth factors,antibodies, antigens and DNA. EVAc matrices allow a high degree ofcontrol over agent release, versatility in allowing the release of awide range of agents, and good retention of biological activity.Biodegradable polymers have also been used to release growth factors,protein hormones, antibodies, antigens and DNA. Biodegradable materialsdisappear from the implant site after protein release. Polymer gelsmight also be useful for topical or localized protein delivery. Systemsthat release multiple protein factors are also possible (Saltzman andOlbricht, Nat Rev Drug Discov, 1:177-86, 2002; Torchilin and Lukyanov,Drug Discov Today, 8:259-66, 2003).

Biodegradable polymers include non-water-soluble polymers that aredegraded by surface or bulk erosion in addition to water-soluble gelsthat dissolve and are cleared from the body without undergoing adecrease in molecular weight. There are many different types ofbiodegradable polymers that can potentially be used in the preparationof peptide delivery systems. They include both naturally derived andsynthetic materials.

Biocompatibility of Polymeric Systems

Polymers used as drug delivery systems for protein pharmaceuticals needto exhibit biocompatible characteristics in terms of both the polymer'seffect on the organism receiving the drug delivery system and thepolymer's effect on the protein to be delivered. Several aspects of apolymeric delivery system ultimately contribute to its overallbiocompatibility, or lack thereof. The polymer itself, which consists ofa repeating monomeric species, may potentially be antigenic,carcinogenic, or toxic or have some inherent incompatibility withorganisms. The shape of an implanted material has been implicated in itsbiocompatibility as well, smooth surfaces being less irritating and morebiocompatible than rough surfaces (Saltzman and Olbricht, Nat Rev DrugDiscov, 1:177-86, 2002).

Pharmaceutical Stability

Interactions between proteins and polymeric materials appear to beprotein and polymer specific. At issue are the protein molecular weight,which is an important parameter with regard to diffusion characteristicsand the iso-electric point of the protein (and polymer as well in somecases), which governs charge-charge interactions (protein-polymer andprotein-protein). Moreover the presence of cysteines on the protein mayfacilitate the formation of intermolecular (i.e., protein-polymer)disulfide bonds. Furthermore, the primary amino acid sequence of theprotein may be rendered susceptible to chemical modification inassociation with a polymeric material. The presence or absence ofcarbohydrates on the protein may enhance or prevent interaction withpolymeric materials and affect the protein's hydrodynamic volume. Therelative hydrophobicity of a protein could interact with hydrophobicsites on a polymer. Finally the heterogeneity of protein pharmaceuticalsoften exists for proteins produced by recombinant methods (Bilati etal., Eur J Pharm Biopharm, 59:375-88, 2005; Gombotz and Pettit,Bioconjug Chem, 6:332-51, 1995; Saltzman and Olbricht, Nat Rev DrugDiscov, 1:177-86, 2002).

Bulk Erosion Polymers Poly(Lactic-Co-Glycolic Acid)

Poly(lactic-co-glycolic acid) (PLGA) has been used successfully forseveral decades in biodegradable structures and more recently as drugdelivery micro-carriers, and as a result of the extended use, much isknown about their biocompatibility and physicochemical characteristics.PLGA copolymers are well suited for use in delivery systems since theycan be fabricated into a variety of morphologies including films, rods,spheres by solvent casting, compression molding and solvent evaporationtechniques. Examples of peptide drug delivery systems made from PLGAcopolymers, have successfully met FDA approval and they are available asmarketed products are Lupron Depot, Zoladex and Decapeptyl (Frokjaer andOtzen, Nat Rev Drug Discov, 4:298-306, 2005).

Block Copolymers of PEG and PLA

Copolymers of PEG and PLA have been synthesized for use in deliverysystems. The net result is a biodegradable polymer with a reduced amountof hydrophobicity that is an inherent property of PLA systems. Thesecopolymer systems can be composed of random blocks of the two polymers,two blocks in which case the molecules are amphiphilic, or triblocks inwhich hydrophilic microphases are present. Peptides that areincorporated into devices made from these copolymers are less likely toadsorb to the delivery system through hydrophobic interactions. Thepolymers were shown to swell very rapidly due to microphase separation,and degradation occurred over 2-3 weeks (Bilati et al., Eur J PharmBiopharm, 59:375-88, 2005; Gombotz and Pettit, Bioconjug Chem, 6:332-51,1995).

Poly(Cyanoacrylates)

Poly(cyanoacrylates) have received attention as delivery systems forproteins and peptides. They undergo spontaneous polymerization at roomtemperature in the presence of water, and their erosion has been shownto be controlled by the length of the monomer chain and the pH. Onceformed, the polymer is slowly hydrolyzed, leading to a chain scissionand liberation of formaldehyde. While the polymers are not toxic, theformaldehyde released as the degradation byproduct does create atoxicity concern. A characteristic example of their use are deliverysystems for insulin prepared by the interfacial emulsion polymerizationof alkyl cyanoacrylate (Gombotz and Pettit, Bioconjug Chem, 6:332-51,1995).

Surface Erosion Polymers Poly(Anhydrides)

Poly(anhydrides) represent a class of surface eroding polymers.Hydrolysis of the anhydride bond is suppressed by acid, which results inan inhibition of bulk erosion by the acidity of the carboxylic acidproducts of the polymer hydrolysis process. By varying the ratio oftheir hydrophobic components, one can control degradation rates rangingfrom days to years. Several proteins have been successfully incorporatedinto, and released, from poly-(anhydride) delivery systems. Theincorporation of insulin and myoglobin has successfully been achieved inpoly(anhydride) microspheres using both a hot-melt microencapsulationtechnique or microencapsulation by solvent removal (Gombotz and Pettit,Bioconjug Chem, 6:332-51, 1995).

Poly(Ortho Esters)

Poly(ortho esters) are another example of surface-eroding polymers thathave been developed for drug delivery systems. Several proteins andpeptides have been incorporated into poly(ortho-ester) delivery systemsincluding the LHRH analog nafarelin, insulin and lysozyme.

Hydrogels

The use of biodegradable hydrogels as delivery systems for proteins isof particular interest due to their biocompatibility and their relativeinertness toward protein drugs (Gombotz and Pettit, Bioconjug Chem,6:332-51, 1995). Hydrogels are the only class of polymer that can enablea protein to permeate through the continuum of the carrier. The initialrelease rate of proteins from biodegradable hydrogels is thereforegenerally diffusion controlled through the aqueous channels of the geland is inversely proportional to the molecular weight of the protein.Once polymer degradation occurs, and if protein still remains in thehydrogel, erosion-controlled release may contribute to the system.Several disadvantages must be considered when using a biodegradablehydrogel system for the release of proteins. Their ability to rapidlyswell with water can lead to very fast release rates and polymerdegradation rates. In addition, hydrogels can rapidly decrease inmechanical strength upon swelling with water. Examples of hydrogelsinclude, pluronic polyols, poly(vinyl alcohol), poly(vinylpyrrolidone),malein anhydride, callulose, hyaluronic acid derivatives, alginate,collagens, gelatin, starches and dextrans.

Selective Drug Delivery

Selective delivery of therapeutic agents includes any methodology bywhich the functional concentration of drug is higher at the target sitethan in normal tissue. A wide variety of methods may fall under thecategory of “selective delivery,” including interventions as simple andmechanical as selective vascular administration in which the drug isphysically isolated in a neovascularized area. An example of that typeof mechanical selectivity is also the EPR effect.

Most strategies, however, are pharmaceutical. In these approaches, thedifferences in the biochemical and physiological nature of normal andthe targeted cells and their microenvironment are exploited forselective delivery. In one embodiment, a carrier is used to deliver apeptide of the invention that because of its physical properties,accumulates preferentially at a target site. In another embodiment, aligand is conjugated to a peptide of the invention that binds to atissue-associated antigen. In another embodiment, an agent of theinvention is maintained in an inactive form that can be activatedpreferentially at the tissue site. In yet another embodiment, externalenergy irradiation is used to release a peptide at the delivery site.

A variety of technologies using combinations of different approaches areconstantly being developed for selective delivery of therapeutics. Thesedelivery systems employ different targets such as cancer cell andneovascular antigens, hypoxia, or high osmotic pressure; targetingagents such as monoclonal antibodies (mAbs), single chain variablefragments (scFvs), peptides and oligonucleotides; effectors likechemical or biological toxins, radioisotopes, genes, enzymes,immunomodulators, oligonucleotides, imaging and diagnostic agents;vehicles the already mentioned colloidal systems, including liposomes,emulsions, micelles, nanoparticles, polymer conjugates or implants; anddrug-releasing switches such as systems that utilize thermal, radiation,ultrasound or magnetic fields (Allen and Cullis, Science, 303:1818-22,2004; Gombotz and Pettit, Bioconjug Chem, 6:332-51, 1995; Moses et al.,Cancer Cell, 4:337-41, 2003; Neri and Bicknell, Nat Rev Cancer,5:436-46, 2005; Saltzman and Olbricht, Nat Rev Drug Discov, 1:177-86,2002).

Tumor Marker Targeting

The advent of aptamer and antibody technology has facilitated the use ofcancer-specific monoclonal antibodies and aptamers to deliver peptidesof the invention to a selected target tissue. Of special interest areantibodies and aptamers that target, in vivo, tumor endothelium. Thosetargets include, but are not limited to, the fibronectin extra-domain B,large tenascin-C isoforms, various integrins, VEGF receptors, prostatespecific membrane antigen, endoglin and CD44 isoforms (Shukla and Krag,Expert Opin Biol Ther, 6:39-54, 2006). Alternatively, the tumor itselfmay be targeted, exemplary tumor markers include CA-125, gangliosidesG(D2), G(M2) and G(D3), CD20, CD52, CD33, Ep-CAM, CEA, bombesin-likepeptides, PSA, HER2/neu, epidermal growth factor receptor, erbB2, erbB3,erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFRs (e.g.,VEGFR3), estrogen receptors, Lewis-Y antigen, TGFβ1, IGF-1 receptor,EGFα, c-Kit receptor, transferrin receptor, IL-2R and CO17-1A. Aptamersand antibodies of the invention can recognize tumors derived from a widevariety of tissue types, including, but not limited to, breast,prostate, colon, lung, pharynx, thyroid, lymphoid, lymphatic, larynx,esophagus, oral mucosa, bladder, stomach, intestine, liver, pancreas,ovary, uterus, cervix, testes, dermis, bone, blood and brain. In thecontext of the present invention, a tumor cell is a neoplastic (e.g.,cancer) cell or a mass of cancer cells, which can also encompass cellsthat support the growth and/or propagation of a cancer cell, such asvasculature and/or stroma, but not necessarily macrophages. Forinstance, therefore, the present invention envisages compositions andmethods for reducing growth of a tumor cell in a subject, whereinantibodies or aptamers bind with specificity to cell surface epitopes(or epitopes of receptor-binding molecules) of a cancer cell or a cellthat is involved in the growth and/or propagation of a cancer cell suchas a cell comprising the vasculature of a tumor or blood vessels thatsupply tumors and/or stromal cells. Methods of this invention areparticularly suitable for administration to humans with neoplasticdiseases.

Antibodies

Antibodies are well known to those of ordinary skill in the science ofimmunology. Particularly useful in the methods of the invention areantibodies that specifically bind a polypeptide that is expressed in atumor or endothelial cell. As used herein, the term “antibody” means notonly intact antibody molecules, but also fragments of antibody moleculesthat retain immunogen binding ability. Such fragments are also wellknown in the art and are regularly employed both in vitro and in vivo.Accordingly, as used herein, the term “antibody” means not only intactimmunoglobulin molecules but also the well-known active fragmentsF(ab′)₂, and Fab. F(ab′)₂, and Fab fragments which lack the Fc fragmentof intact antibody, clear more rapidly from the circulation, and mayhave less non-specific tissue binding of an intact antibody (Wahl etal., J. Nucl. Med. 24:316-325, 1983). The antibodies of the inventioncomprise whole native antibodies, bispecific antibodies; chimericantibodies; Fab, Fab′, single chain V region fragments (scFv) and fusionpolypeptides.

In one embodiment, an antibody that binds polypeptide is monoclonal.Alternatively, the antibody is a polyclonal antibody. The preparationand use of polyclonal antibodies are also known to the skilled artisan.The invention also encompasses hybrid antibodies, in which one pair ofheavy and light chains is obtained from a first antibody, while theother pair of heavy and light chains is obtained from a different secondantibody. Such hybrids may also be formed using humanized heavy andlight chains. Such antibodies are often referred to as “chimeric”antibodies.

In general, intact antibodies are said to contain “Fc” and “Fab”regions. The Fc regions are involved in complement activation and arenot involved in antigen binding. An antibody from which the Fc′ regionhas been enzymatically cleaved, or which has been produced without theFc′ region, designated an “F(ab′)₂” fragment, retains both of theantigen binding sites of the intact antibody. Similarly, an antibodyfrom which the Fc region has been enzymatically cleaved, or which hasbeen produced without the Fc region, designated an “Fab′” fragment,retains one of the antigen binding sites of the intact antibody. Fab′fragments consist of a covalently bound antibody light chain and aportion of the antibody heavy chain, denoted “Fd.” The Fd fragments arethe major determinants of antibody specificity (a single Fd fragment maybe associated with up to ten different light chains without alteringantibody specificity). Isolated Fd fragments retain the ability tospecifically bind to immunogenic epitopes.

Antibodies can be made by any of the methods known in the art utilizingan HSP27 polypeptide or a polypeptide described in Table 1, orimmunogenic fragments thereof, as an immunogen. One method of obtainingantibodies is to immunize suitable host animals with an immunogen and tofollow standard procedures for polyclonal or monoclonal antibodyproduction. The immunogen will facilitate presentation of the immunogenon the cell surface. Immunization of a suitable host can be carried outin a number of ways. Nucleic acid sequences encoding an HSP27polypeptide or a polypeptide described in Table 1, or immunogenicfragments thereof, can be provided to the host in a delivery vehiclethat is taken up by immune cells of the host. The cells will in turnexpress the receptor on the cell surface generating an immunogenicresponse in the host. Alternatively, nucleic acid sequences encoding anan HSP27 polypeptide or a polypeptide described in Table 1, orimmunogenic fragments thereof, can be expressed in cells in vitro,followed by isolation of the polypeptide and administration of thereceptor to a suitable host in which antibodies are raised.

Using either approach, antibodies can then be purified from the host.Antibody purification methods may include salt precipitation (forexample, with ammonium sulfate), ion exchange chromatography (forexample, on a cationic or anionic exchange column preferably run atneutral pH and eluted with step gradients of increasing ionic strength),gel filtration chromatography (including gel filtration HPLC), andchromatography on affinity resins such as protein A, protein G,hydroxyapatite, and anti-immunoglobulin.

Antibodies can be conveniently produced from hybridoma cells engineeredto express the antibody. Methods of making hybridomas are well known inthe art. The hybridoma cells can be cultured in a suitable medium, andspent medium can be used as an antibody source. Polynucleotides encodingthe antibody of interest can in turn be obtained from the hybridoma thatproduces the antibody, and then the antibody may be producedsynthetically or recombinantly from these DNA sequences. For theproduction of large amounts of antibody, it is generally more convenientto obtain an ascites fluid. The method of raising ascites generallycomprises injecting hybridoma cells into an immunologically naivehistocompatible or immunotolerant mammal, especially a mouse. The mammalmay be primed for ascites production by prior administration of asuitable composition; e.g., Pristane.

Monoclonal antibodies (Mabs) produced by methods of the invention can be“humanized” by methods known in the art. “Humanized” antibodies areantibodies in which at least part of the sequence has been altered fromits initial form to render it more like human immunoglobulins.Techniques to humanize antibodies are particularly useful when non-humananimal (e.g., murine) antibodies are generated. Examples of methods forhumanizing a murine antibody are provided in U.S. Pat. Nos. 4,816,567,5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.

Aptamers

Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA)ligands that function by folding into a specific globular structure thatdictates binding to target proteins or other molecules with highaffinity and specificity, as described by Osborne et al., Curr. Opin.Chem. Biol. 1:5-9, 1997; and Cerchia et al., FEBS Letters 528:12-16,2002. By “aptamer” is meant a single-stranded polynucleotide that bindsto a protein. Desirably, the aptamers are small, approximately ˜15 KD.The aptamers are isolated from libraries consisting of some 10¹⁴-10¹⁵random oligonucleotide sequences by a procedure termed SELEX (systematicevolution of ligands by exponential enrichment). See Tuerk et al.,Science, 249:505-510, 1990; Green et al., Methods Enzymology. 75-86,1991; Gold et al., Annu. Rev. Biochem., 64: 763-797, 1995; Uphoff etal., Curr. Opin. Struct. Biol., 6: 281-288, 1996. Methods of generatingaptamers are known in the art and are described, for example, in U.S.Pat. Nos. 6,344,318, 6,331,398, 6,110,900, 5,817,785, 5,756,291,5,696,249, 5,670,637, 5,637,461, 5,595,877, 5,527,894, 5,496,938,5,475,096, 5,270,163, and in U.S. Patent Application Publication Nos.20040241731, 20030198989, 20030157487, and 20020172962.

An aptamer of the invention is capable of binding with specificity to apolypeptide expressed by a cell of interest (e.g., a tumor cell or anendothelial cell supplying a tumor). “Binding with specificity” meansthat non-tumor polypeptides are either not specifically bound by theaptamer or are only poorly bound by the aptamer. In general, aptamerstypically have binding constants in the picomolar range. Particularlyuseful in the methods of the invention are aptamers having apparentdissociation constants of 1, 10, 15, 25, 50, 75, or 100 nM.

In one embodiment, an antigen expressed on a blood vessel supplying atumor is the molecular target of the aptamer. Because aptamers can actas direct antagonists of the biological function of proteins, aptamersthat target such polypeptide can be used to modulate angiogenesis,vasculogenesis, blood vessel stabilization or remodeling. Thetherapeutic benefit of such aptamers derives primarily from thebiological antagonism caused by aptamer binding.

The invention encompasses stabilized aptamers having modifications thatprotect against 3′ and 5′ exonucleases as well as endonucleases. Suchmodifications desirably maintain target affinity while increasingaptamer stability in vivo. In various embodiments, aptamers of theinvention include chemical substitutions at the ribose and/or phosphateand/or base positions of a given nucleobase sequence. For example,aptamers of the invention include chemical modifications at the 2′position of the ribose moiety, circularization of the aptamer, 3′capping and ‘spiegelmer’ technology. Aptamers having A and G nucleotidessequentially replaced with their 2′-OCH3 modified counterparts areparticularly useful in the methods of the invention. Such modificationsare typically well tolerated in terms of retaining aptamer affinity andspecificity. In various embodiments, aptamers include at least 10%, 25%,50%, or 75% modified nucleotides. In other embodiments, as many as80-90% of the aptamer's nucleotides contain stabilizing substitutions.In other embodiments, 2′-OMe aptamers are synthesized. Such aptamers aredesirable because they are inexpensive to synthesize and naturalpolymerases do not accept 2′-OMe nucleotide triphosphates as substratesso that 2′-OMe nucleotides cannot be recycled into host DNA. A fully2′-O-methyl aptamer, named ARC245, was reported to be so stable thatdegradation could not be detected after 96 hours in plasma at 37° C. orafter autoclaving at 125° C. Using methods described herein, aptamerswill be selected for reduced size and increased stability. In oneembodiment, aptamers having 2′-F and 2′-OCH₃ modifications are used togenerate nuclease resistant aptamers. Other modifications that stabilizeaptamers are known in the art and are described, for example, in U.S.Pat. No. 5,580,737; and in U.S. Patent Application Publication Nos.20050037394, 20040253679, 20040197804, and 20040180360.

Using standard methods tumor markers or endothelial call-specificaptamers can be selected that bind virtually any tumor marker orendothelial cell-expressed polypeptide known in the art.

The Fibronectin Extra-Domain B (EDB)

Fibronectin is a large glycoprotein that is present in large amounts inthe plasma and tissues. EDB is a 91-amino-acid type III homology domainthat becomes inserted into the fibronectin molecule undertissue-remodeling conditions by a mechanism of alternative splicing atthe level of the primary transcript. EDB is essentially undetectable inhealthy adult individuals. EDB-containing fibronectin is abundant inmany aggressive solid tumors and in neo-vascularized endothelium, anddisplays either predominantly vascular or diffuse stromal patterns ofexpression, depending on the tissue.

Large Tenascin-C Isoforms

Tenascins are a family of four extracellular matrix glycoproteins thatare found in vertebrates. They are typically present in many differentconnective tissues. Tenascins contribute to matrix structure andinfluence the behavior of cells that are in contact with theextracellular matrix. Several isoforms of tenascin-C can be generated asa result of different patterns of alternative splicing in the regionbetween domains A1 and D. It has been known for some time that splicedisoforms containing extra domains are tumor-associated antigens, whichshow a more restricted pattern of expression in normal tissues comparedwith the “small” tenascin isoforms. The C domain of tenascin-C is theextra domain that shows the most restricted pattern of expression. Innormal adult tissue it is undetectable by immunohistochemistry andnorthern-blot analysis, but it is strongly expressed in aggressive braintumors and some lung tumors, with a prominent perivascular pattern ofstaining.

Integrins

During vascular remodeling and angiogenesis, endothelial cells showincreased expression of several cell-surface molecules that potentiatecell invasion and proliferation. One such molecule is the integrinαv-β3, which has a key role in endothelial cell survival duringangiogenesis in vivo and which might serve as a target for therapeuticmolecules, particularly those that require internalization inendothelial cells. Monoclonal antibodies to the αv-β3 have been shown todisplay anti-angiogenic activities and to preferentially stain tumorblood vessels.

VEGFs and Their Receptors

VEGFs represent a class of proteins that promote angiogenesis, increasevascular permeability and contribute to endothelial-cell survival inblood and lymphatic vessels. The contribution of VEGFA to cancerprogression has been highlighted by the recent approval of the humanizedanti-VEGF monoclonal antibody bevacizumab (Avastin; Genentech) forfirst-line cancer treatment. The overexpression of VEGFs and VEGFreceptors in tumors is well documented. The selective tumor localizationof monoclonal antibodies to VEGFA, VEGF receptor 2 and the VEGFA-VEGFreceptor 2 complex can be used as an excellent selectivity mechanism fortargeting the angiogenic vasculature.

Prostate-Specific Membrane Antigen

Prostate-specific membrane antigen (PSMA) is a membrane glycoproteinwith proteolytic activity. PSMA is predominantly expressed in theprostate and serum concentrations are often increased in patients withprostate cancer. Several studies have reported overexpression of PSMA inthe neo-vasculature of different solid tumors, whereas expression innormal vasculature is limited to some vessels of the breast, duodenum,kidney and prostate.

Endoglin

Endoglin (CD105) is a transforming growth factor-beta (TGF) co-receptorthat is overexpressed in tumor neo-vasculature and is used as a markerfor the tumor endothelium.

CD44 Isoforms

CD44 is a cell-surface receptor of great molecular heterogeneity, whichis due to both alternative splicing and extensive post-translationalmodification. The radio-labeled monoclonal antibody TES-23, which isspecific to an isoform of CD44, has shown impressive performance intumor-targeting experiments in animal models. TES-23 recognizes a widelydistributed form of CD44 that lacks variant exons, known as CD44H.

Tumor Endothelial Markers (TEMs)

TEMs is a family of genes encoding proteins that serve as tumorendothelial markers (Carson-Walter, Watkins, et al, Cancer Res.61:6649-55, 2001). These genes display elevated expression during tumorangiogenesis. From both biological and clinical points of view, TEMsassociated with the cell surface membrane are of particular interest.Accordingly, four such genes are characterized, TEM1, TEM5, TEM7, andTEM8, all of which contain putative transmembrane domains. TEM5 appearsto be a seven-pass transmembrane receptor, whereas TEM1, TEM7, and TEM8span the membrane once. Three of these TEMs (TEM1, TEM5, and TEM8) areabundantly expressed in tumor vessels in mouse tumors, embryos, andadult tissues as well as in the vasculature of the developing mouseembryo. The expression of these TEMs in normal adult mice tissues isundetectable.

Selective Delivery Through Pro-Drug Activation

Selective delivery of agents of the invention can be achieved byadministering a pro-drug form that is converted into an active drug atthe target site. Most pro-drugs are designed to have a “trigger,”“linker” and “effector.” The “trigger,” following the tissue-specificmetabolism, modifies the “linker,” resulting in an activation of the“effector.” There are several mechanisms potentially exploitable forselective activation. Some utilize unique aspects of the tissuephysiology, such as selective enzyme expression or hypoxia in the caseof tumors, whereas others are based on tissue antigen-specific deliverytechniques.

Targeting Secreted Enzymes from Cells

The approach uses pro-drugs that are “hidden” from the cells untilcleaved by an enzyme produced and secreted preferentially by the cells.A typical example of an enzyme used for pro-drug activation is MMP-9.

Targeting Tumor Hypoxia

Advances in the chemistry of bio-reductive drug activation have led tothe design of hypoxia-selective drug delivery systems. These pro-drugsinitially undergo one-electron reduction by reductases to give theradical anion, which in normal cells are re-oxidized to the parentcompound, but in hypoxic tumor cells they are further reduced to morehydrophilic species and trapped inside. These drugs can be selectivelydelivered to tumors with defined hypoxic fractions rich in the requiredactivating enzymes.

Antibody-Directed Enzyme Pro-Drug Therapy

Antibody-directed pro-drug therapy (ADEPT) is a 2-step approach in whichfirst the antibody-enzyme construct is administered intravenously. Thisis composed of an antibody against a tissue-specific target linked to anenzyme that activates a pro-drug. In the second step, after the unboundantibody-enzyme conjugate construct is cleared from the circulation, apro-drug is administered intravenously. The pro-drug is an agent thathas been rendered less active by chemical addition of enzyme-cleavablemoieties. The pro-drug is converted to an active form by the tumor-boundantibody-enzyme, which results in local accumulation of the fully activeform of the agent.

External Energy-Controlled Delivery

Some selective delivery strategies involve focusing external energy forconcentrating or delivering therapeutics at the tissue site. A varietyof delivery systems in this category are in the experimental stage,although some have been used in clinical trials as well. Thosestrategies include selective delivery through photodynamic therapy,magnetically targeted delivery, selective delivery through X-rayexposure, radiation-induced selective delivery and ultrasound-guideddelivery.

Methods of Ocular Delivery

The compositions of the invention (e.g., peptides listed in Table 1) arealso particularly suitable for treating ocular diseases, such asage-related macular degeneration, choroidal neovascularization,persistent hyperplastic vitreous syndrome, diabetic retinopathy, andretinopathy of prematurity that are characterized by excessangiogenesis.

In one approach, the compositions of the invention are administeredthrough an ocular device suitable for direct implantation into thevitreous of the eye. The compositions of the invention may be providedin sustained release compositions, such as those described in, forexample, U.S. Pat. Nos. 5,672,659 and 5,595,760. Such devices are foundto provide sustained controlled release of various compositions to treatthe eye without risk of detrimental local and systemic side effects. Anobject of the present ocular method of delivery is to maximize theamount of drug contained in an intraocular device or implant whileminimizing its size in order to prolong the duration of the implant.See, e.g., U.S. Pat. Nos. 5,378,475; 6,375,972, and 6,756,058 and U.S.Publications 20050096290 and 200501269448. Such implants may bebiodegradable and/or biocompatible implants, or may be non-biodegradableimplants. Biodegradable ocular implants are described, for example, inU.S. Patent Publication No. 20050048099. The implants may be permeableor impermeable to the active agent, and may be inserted into a chamberof the eye, such as the anterior or posterior chambers or may beimplanted in the schlera, transchoroidal space, or an avascularizedregion exterior to the vitreous. Alternatively, a contact lens that actsas a depot for compositions of the invention may also be used for drugdelivery.

In a preferred embodiment, the implant may be positioned over anavascular region, such as on the sclera, so as to allow for transcleraldiffusion of the drug to the desired site of treatment, e.g. theintraocular space and macula of the eye. Furthermore, the site oftranscleral diffusion is preferably in proximity to the macula. Examplesof implants for delivery of a composition include, but are not limitedto, the devices described in U.S. Pat. Nos. 3,416,530; 3,828,777;4,014,335; 4,300,557; 4,327,725; 4,853,224; 4,946,450; 4,997,652;5,147,647; 5,164,188; 5,178,635; 5,300,114; 5,322,691; 5,403,901;5,443,505; 5,466,466; 5,476,511; 5,516,522; 5,632,984; 5,679,666;5,710,165; 5,725,493; 5,743,274; 5,766,242; 5,766,619; 5,770,592;5,773,019; 5,824,072; 5,824,073; 5,830,173; 5,836,935; 5,869,079,5,902,598; 5,904,144; 5,916,584; 6,001,386; 6,074,661; 6,110,485;6,126,687; 6,146,366; 6,251,090; and 6,299,895, and in WO 01/30323 andWO 01/28474, all of which are incorporated herein by reference.

Examples include, but are not limited to the following: a sustainedrelease drug delivery system comprising an inner reservoir comprising aneffective amount of an agent effective in obtaining a desired local orsystemic physiological or pharmacological effect, an inner tubeimpermeable to the passage of the agent, the inner tube having first andsecond ends and covering at least a portion of the inner reservoir, theinner tube sized and formed of a material so that the inner tube iscapable of supporting its own weight, an impermeable member positionedat the inner tube first end, the impermeable member preventing passageof the agent out of the reservoir through the inner tube first end, anda permeable member positioned at the inner tube second end, thepermeable member allowing diffusion of the agent out of the reservoirthrough the inner tube second end; a method for administering a compoundof the invention to a segment of an eye, the method comprising the stepof implanting a sustained release device to deliver the compound of theinvention to the vitreous of the eye or an implantable, sustainedrelease device for administering a compound of the invention to asegment of an eye; a sustained release drug delivery device comprising:a) a drug core comprising a therapeutically effective amount of at leastone first agent effective in obtaining a diagnostic effect or effectivein obtaining a desired local or systemic physiological orpharmacological effect; b) at least one unitary cup essentiallyimpermeable to the passage of the agent that surrounds and defines aninternal compartment to accept the drug core, the unitary cup comprisingan open top end with at least one recessed groove around at least someportion of the open top end of the unitary cup; c) a permeable plugwhich is permeable to the passage of the agent, the permeable plug ispositioned at the open top end of the unitary cup wherein the grooveinteracts with the permeable plug holding it in position and closing theopen top end, the permeable plug allowing passage of the agent out ofthe drug core, through the permeable plug, and out the open top end ofthe unitary cup; and d) at least one second agent effective in obtaininga diagnostic effect or effective in obtaining a desired local orsystemic physiological or pharmacological effect; or a sustained releasedrug delivery device comprising: an inner core comprising an effectiveamount of an agent having a desired solubility and a polymer coatinglayer, the polymer layer being permeable to the agent, wherein thepolymer coating layer completely covers the inner core.

Other approaches for ocular delivery include the use of liposomes totarget a compound of the present invention to the eye, and preferably toretinal pigment epithelial cells and/or Bruch's membrane. For example,the compound may be complexed with liposomes in the manner describedabove, and this compound/liposome complex injected into patients with anocular disease, using intravenous injection to direct the compound tothe desired ocular tissue or cell. Directly injecting the liposomecomplex into the proximity of the retinal pigment epithelial cells orBruch's membrane can also provide for targeting of the complex with someforms of ocular PCD. In a specific embodiment, the compound isadministered via intra-ocular sustained delivery (such as VITRASERT orENVISION). In a specific embodiment, the compound is delivered byposterior subtenons injection. In another specific embodiment,microemulsion particles containing the compositions of the invention aredelivered to ocular tissue to take up lipid from Bruch's membrane,retinal pigment epithelial cells, or both.

For optical applications, nanoparticles are a colloidal carrier systemthat has been shown to improve the efficacy of the encapsulated drug byprolonging the serum half-life. Polyalkylcyanoacrylates (PACAs)nanoparticles are a polymer colloidal drug delivery system that is inclinical development, as described by Stella et al., J. Pharm. Sci.,2000. 89: p. 1452-1464; Brigger et al., Int. J. Pharm., 2001. 214: p.37-42; Calvo et al., Pharm. Res., 2001. 18: p. 1157-1166; and Li et al.,Biol. Pharm. Bull., 2001. 24: p. 662-665. Biodegradable poly (hydroxylacids), such as the copolymers of poly (lactic acid) (PLA) and poly(lactic-co-glycolide) (PLGA) are being extensively used in biomedicalapplications and have received FDA approval for certain clinicalapplications. In addition, PEG-PLGA nanoparticles have many desirablecarrier features including (i) that the agent to be encapsulatedcomprises a reasonably high weight fraction (loading) of the totalcarrier system; (ii) that the amount of agent used in the first step ofthe encapsulation process is incorporated into the final carrier(entrapment efficiency) at a reasonably high level; (iii) that thecarrier have the ability to be freeze-dried and reconstituted insolution without aggregation; (iv) that the carrier be biodegradable;(v) that the carrier system be of small size; and (vi) that the carrierenhance the particles persistence.

Nanoparticles are synthesized using virtually any biodegradable shellknown in the art. In one embodiment, a polymer, such as poly(lactic-acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) is used.Such polymers are biocompatible and biodegradable, and are subject tomodifications that desirably increase the photochemical efficacy andcirculation lifetime of the nanoparticle. In one embodiment, the polymeris modified with a terminal carboxylic acid group (COOH) that increasesthe negative charge of the particle and thus limits the interaction withnegatively charge nucleic acid aptamers. Nanoparticles are also modifiedwith polyethylene glycol (PEG), which also increases the half-life andstability of the particles in circulation. Alternatively, the COOH groupis converted to an N-hydroxysuccinimide (NHS) ester for covalentconjugation to amine-modified aptamers.

Biocompatible polymers useful in the composition and methods of theinvention include, but are not limited to, polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetage phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulphate sodium salt,poly(methyl methacrylate), poly(ethylmethacrylate),poly(butylmethacrylate), poly(isobutylmethacryla-te),poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), poly(vinyl acetate, poly vinyl chloride polystyrene,polyvinylpryrrolidone, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodeclmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecl acrylate) and combinations of any of these. Inone embodiment, the nanoparticles of the invention include PEG-PLGApolymers.

Compositions of the invention may also be delivered topically. Fortopical delivery, the compositions are provided in any pharmaceuticallyacceptable excipient that is approved for ocular delivery. Preferably,the composition is delivered in drop form to the surface of the eye. Forsome application, the delivery of the composition relies on thediffusion of the compounds through the cornea to the interior of theeye.

Those of skill in the art will recognize that the best treatmentregimens for using compounds of the present invention to treat an ocularPCD can be straightforwardly determined. This is not a question ofexperimentation, but rather one of optimization, which is routinelyconducted in the medical arts. In vivo studies in nude mice oftenprovide a starting point from which to begin to optimize the dosage anddelivery regimes. The frequency of injection will initially be once aweek, as has been done in some mice studies. However, this frequencymight be optimally adjusted from one day to every two weeks to monthly,depending upon the results obtained from the initial clinical trials andthe needs of a particular patient.

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice, as a skilled artisan recognizes itis routine in the art to modify the dosage for humans compared to animalmodels. In certain embodiments it is envisioned that the dosage may varyfrom between about 1 mg compound/Kg body weight to about 5000 mgcompound/Kg body weight; or from about 5 mg/Kg body weight to about 4000mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kgbody weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg bodyweight; or from about 100 mg/Kg body weight to about 1000 mg/Kg bodyweight; or from about 150 mg/Kg body weight to about 500 mg/Kg bodyweight. In other embodiments this dose may be about 1, 5, 10, 25, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, 5000 mg/Kg body weight. In other embodiments, it is envisaged thathigher doses may be used, such doses may be in the range of about 5 mgcompound/Kg body to about 20 mg compound/Kg body. In other embodimentsthe doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Ofcourse, this dosage amount may be adjusted upward or downward, as isroutinely done in such treatment protocols, depending on the results ofthe initial clinical trials and the needs of a particular patient.

Combination Therapies

Optionally, an angiogenic modulating therapeutic as described herein maybe administered in combination with any other standard active angiogenicmodulating therapeutics; such methods are known to the skilled artisanand described in Remington's Pharmaceutical Sciences by E. W. Martin.For example, an anti-angiogenic peptide of the invention may beadministered in combination with any other anti-angiogenic peptide, orwith known anti-angiogenic agent. Such agents are listed below (Folkman,Annu Rev Med. 57:1-18, 2006).

Agent Clinical Trials  1. Alphastatin  2. Angiostatin  3. Arresten  4.Anti-thrombin III (truncated)  5. Canstatin  6. Endostatin Phase II  7.Fibulin-5  8. Fragment of histidine-rich glycoprotein  9. Interferon-βPhase III 10. Maspin 11. 2-methoxyestradiol Phase II 12. PEX 13. Pigmentepithelial-derived factor (PEDF) 14. Platelet factor 4 (PF4) 15.Semaphorin 3F 16. sFlt-1 17. Tetrahydrocortisol Phase III 18.Thrombospondin-1 (and -2) Phase II 19. TIMP-2 20. Troponin I 21.Tumstatin 22. Vasostatin

For the treatment of a neoplasia, a peptide of the invention (e.g., anyone or more of those listed in Table 1) is administered in combinationwith any conventional treatment (e.g., chemotherapy, radiotherapy,hormonal therapy, surgery, cryosurgery). A pharmaceutical composition ofthe invention may, if desired, include one or more chemotherapeuticstypically used in the treatment of a neoplasm, such as abirateroneacetate, altretamine, anhydrovinblastine, auristatin, bexarotene,bicalutamide,BMS184476,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, bleomycin,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide,cachectin, cemadotin, chlorambucil, cyclophosphamide,3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine, docetaxol,doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNU),cisplatin,cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC),dactinomycin, daunorubicin, dolastatin, doxorubicin (adriamycin),etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea andhydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU),mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate,rhizoxin, sertenef, streptozocin, mitomycin, methotrexate,5-fluorouracil, nilutamide, onapristone, paclitaxel, prednimustine,procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin,taxol, thalidomide, tretinoin, vinblastine, vincristine, vindesinesulfate, and vinflunine. Other examples of chemotherapeutic agents canbe found in Cancer Principles and Practice of Oncology by V. T. Devitaand S. Hellman (editors), 6th edition (Feb. 15, 2001), LippincottWilliams & Wilkins Publishers.

Kits

The invention provides kits for the treatment or prevention of diseasesor disorders characterized by excess or undesirable angiogenesis. In oneembodiment, the kit includes a therapeutic or prophylactic compositioncontaining an effective amount of one or more peptides of Table 1 (SEQID Nos. 1-156) in unit dosage form. In some embodiments, the kitcomprises a sterile container that contains a therapeutic orprophylactic vaccine; such containers can be boxes, ampoules, bottles,vials, tubes, bags, pouches, blister-packs, or other suitable containerforms known in the art. Such containers can be made of plastic, glass,laminated paper, metal foil, or other materials suitable for holdingmedicaments.

If desired a peptide of the invention is provided together withinstructions for administering it to a subject having or at risk ofdeveloping excess or undesired angiogenesis. The instructions willgenerally include information about the use of the composition for thetreatment or prevention of ischemia or for enhancing angiogenesis to atissue in need thereof. In other embodiments, the instructions includeat least one of the following: description of the expression vector;dosage schedule and administration for treatment or prevention ofischemia or symptoms thereof; precautions; warnings; indications;counter-indications; overdosage information; adverse reactions; animalpharmacology; clinical studies; and/or references. The instructions maybe printed directly on the container (when present), or as a labelapplied to the container, or as a separate sheet, pamphlet, card, orfolder supplied in or with the container.

Methods of the Invention

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Handbook of Experimental Immunology” (Weir, 1996);“Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);“Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: ThePolymerase Chain Reaction”, (Mullis, 1994); “Current Protocols inImmunology” (Coligan, 1991). These techniques are applicable to theproduction of the polynucleotides and polypeptides of the invention,and, as such, may be considered in making and practicing the invention.Particularly useful techniques for particular embodiments will bediscussed in the sections that follow.

EXAMPLES Example 1 Bioinformatics Approach to Identifying AngiogenesisInhibitors

During the last two decades, a large number of endogenous regulatorshave been identified that either stimulate or inhibit the process ofangiogenesis. Disturbance of the fine regulation of these stimulatingand inhibiting elements leads to pathologic conditions. One of thehallmarks of cancer progression is the shift of these regulatoryelements towards the pro-angiogenic components, often referred to as theangiogenic switch (Folkman, Semin Oncol, 29:15-8, 2002).

The angiogenesis-promoting regulatory elements include various growthfactors. Growth factor signaling promotes the expression ofextracellular matrix (ECM)-processing enzymes such as the freelydiffusing and membrane-bound matrix metalloproteinases (MMPs), plasmin,and various serine, cysteine and aspartic acid proteinases (cathepsins).While the importance of these enzymes, particularly the cell-secretedproteases, in processing the extracellular matrix has been demonstratedboth experimentally (Neri and Bicknell, Nat Rev Cancer, 5:436-46, 2005)and theoretically (Karagiannis and Popel, J Biol Chem, 279:39105-14,2004), there are indications that this role may not be unique. Onlyrecently has it been realized that the proteolytic processing of the ECMcomponents results in the exposure of biologically active proteinfragments, known as cryptic fragments (Davis et al., Am J Pathol,156:1489-98, 2000). These fragments are fully functional moieties orinclude active sites within larger sequences that are normally hiddenfrom the extracellular environment inside the structure of the ECMmacromolecules. Proteolytic processing of the macromolecules revealsthese fragments that, once released, can act as modulators ofangiogenesis. Most of these fragments have anti-angiogenic andpro-apoptotic activities; they act as endogenous inhibitors ofangiogenesis. Others have pro-angiogenic properties. The presentinvention is focused on angiogenesis inhibitors.

The amino-acid sequences of the known potent anti-angiogenic fragmentswere compiled after extensive searching of the relevant literature. Intotal 36 proteins with known anti-angiogenic and pro-apoptoticactivities were found (Table 2).

TABLE 2 36 target sequences with anti-angiogenic or pro-apoptoticproperties. Length of known active domain 20-60 aa 100-150 aa >150 aaCandidate Endorepellin/LG3 Angiostatin K5 ADAMTS-1 antiangiogenicKininogen frg BCA-1 Angiostatin proteins or K1-4 protein PF-4 EndostatinAngio- fragments tensinogen Timp2/loop 6 Gro-β Antithrombin TSP1/Mal-IIGrowth Hormone Arresten TSP1/Mal-III IP-10 BAI-1 Tumstatin/Tum2 I-TACBAI-2 Tumstatin/Tum3 Kininogen/D5 BAI-3 Tumstatin/Tum4 LactogenCalreticulin Tumstatin/Tum7 MIG Canstatin Prolactin Kallistatin MaspinPEDF Restin VastatinThe proteins are categorized according to the information known aboutthe length of the amino acid sequence within which their activity islocalized. The proteins are listed alphabetically.

Based on the information inferred from the amino acid sequences of theknown anti-angiogenic proteins, these proteins were systematicallycategorized by protein family. After observing that most of the knownanti-angiogenic active domains were localized within conserved proteindomains, the proteins were classified according to these conserveddomains. Along with this classification, the information from the aminoacid sequences of the active fragments was used to evaluate sequencesimilarities and predict novel proteins that likely induceanti-angiogenic effects.

These proteins were divided into three classes by length of known activefragment (i.e., 20-60 amino acids; 100-150 amino acids; or greater than150 amino acids) according to the known information regarding the activefragment and the localization of their anti-angiogenic activity. Some ofthese thirty-six proteins have known “active domains,” which have up toapproximately 20 amino acids, that are known to exhibit potentanti-angiogenic activity. Others are protein fragments of 100-150 aminoacids with identified activity but an unknown active domain, and stillothers are full-length proteins or large protein domains, having morethan 150 contiguous amino acid sequences, that are known to be potentangiogenesis inhibitors.

The top 202 identified similarity hits for the proteins or proteinfragments with known anti-angiogenic activity are displayed in Table 3.Of the 202 hits, some of which are duplicated among the queries, over150 distinct novel anti-angiogenic protein segments. The queries thatinclude the proteins from Table 2 are displayed in the first column andorganized alphabetically. Among the Table 2 proteins, those withidentified similarities and lengths of 20-60 amino acids were plateletfactor-4, with 7 similar protein domains; various fragments ofthrombospondin 1, each of which with 45 identified similar fragments;and various tumstatins, with approximately 5 similar fragments each. Theproteins with identified similarities and lengths of 100-150 amino acidsincluded the angiostatin kringles; the CXC chemokines Gro-β, IP-10, andMIG, each with approximately 8 top similar protein sites; growthhormone-1 and placental lactogen with 3 similar fragments each; as wellas kininogen, with a single identified similarity. The hits aredisplayed along with their identification numbers and the correspondingpart of their sequence that is similar. They are organized in threecolumns according to the degree of similarity with the query, indescending order of similarity. The similarity notations “identical,”“highly similar,” and “similar” correspond to the scaled score: Proteinswith a score of 80-100% were “identical,” 60-80% “highly similar” and45-60% “similar.”

TABLE 3 Sequences identified by similarity to pro-angiogenicpolypeptides. Identical Highly Similar Similar PF-4 GCP-2/CXCL5(AAH13744.1: 86-109) GRO-β/CXCL2(AAH15753.1: 80-103) ENA-78/CXCL5(AAP35453.1: 55-108) GRO-γ/MIP-28/CXCL3 (AAAB3184.1: 79-100 GRO-α/CXCL1(AAP35528.1: 80-103) IL-B/CXCL8 (AAP35730.1: 72-94) THBG-β/CXCL7(AAB46877.1: 100-121) Timp2/ TIMP 4 (AAV38433.1: 175-198) TIMP 3(AAA21815.1: 148-171) loop 6 TSP1/ T8P-2 (CAI23645.1: THSD1 (AAQ88516.1:347-365) Properdin (AAP43692.1: 143-161) Mal-II 444-462) WISP-1(AAH74841.1: 221-238) ADAMTS-9 (NP891550.1: 585-613) BAI-2 (Q60241:304-322) ADAMTS-10 (NP112219.2:

54-572) ADAMTS-16 (Q8TE57: 1133-1149) ADAMTS-14 (CAI13857.1: 980-994)CILP (AAQ89263.1: 156-175) ADAMTS-13 (AAQ88485.1: 7

1-785) VSGP/F-

pondin (BA818461.1:

21-

39) Papilin (AAH42057.1: 33-

1) BAI-3 (CAI21873.1: 852-870) ADAMTS-1 (Q9UH16: 566-584) ADAMTS-18(AAH63288.1: 1131-1146) WISP-2 (AAQ88274.1: 199-216) ADAMTS-2(CAA05660.1: 952-988) ADAMTS-4 (CAH72148.1: 527-540) ADAMTS-3(NP055058.1: 973-989) ADAMTS-5 (NPP922932.2: 847-880) ADAMTS-9 (O9P2N4:1247-1281) ADAMTS-8 (BAD92954.1: 62-7

) Semaphorin 5A (NP00587.1: 846-86

) ADAMTS-20 (CAD56180.2: 664-681) TSRC1 (AAH27478.1: 140-159) Semaphorin5B (AAO86491.1: 916-934) Fibulin-5 (CAC37890.1: 1688-1706) WISP-3(CAB16556.1: 191-206) CYR51 (AAR05446.1: 234-251) THSD6 (AAH40620.1:44-60) ADAMTS-19 (CAC84565.1: 1096-1111) THSD5 (AAH33140.1: 260-298)ADAMTS-12 (CAC20419.1: 549-582) NOVN (AAL92490.1: 211-228) ADAMTS-20(CAD56150.1: 1661-1675) C8 (AAB69483.1: 30-48) CTGF (CAC44023.1:204-221) UNC5D (AA

88514.1: 259-277) ADAMTS-18 (NP620685.2: 997-1013) CILP-2 (AAN17828.1:153-171) ADAMTS-7 (AAH61631.1: 828-841) ADAMTS-15 (CAC86014.1: 900-916)ADAMTS-5 (NP005969.1: 852-896) UNC5C (AAH41167.1: 267-285) TSP1/ADAMTS-like 3 (NP_997400.1: 425-442) Semaphorin 5B (AAQ38491.1: 731-747)Mal-III Fibulin-5 (CAC37630.1: 1574-1592) VSGP/F-spondin (BA815461.1:821-839) ADAMTS-16 (AAH83283.1: 1131-1147) UNC5C (AAH41168.1: 267-285)BAI-2 (O60241: 304-322) TSRC1 (AAH71852.1: 140-168) TSP-2 (CAI23645.1:501-518) Properdin (AAP43682.1: 143-161) SCO-spondin (XP379957.2:3781-3789) ADAMTS-20 (CAD58160.2: 1309-1326) ADAMTS-16 (QBTE57:1133-1150) ADAMTS-7 (Q9UKP4: 545-563) ADAMTS-12 (NP112217.2: 1480-1495)WISP-1 (AAH74841.1: 221-237) BAI-1 (O14514: 361-379) ADAMTS-15 (QBTE58:848-863) ADAMTS-like 1 (NP443098.2:

3-400) THSD3 (AAI01020.1: 333-351) ADAMTS-16 (CAC86016.1: 5

3-

11) ADAMTS-17 (Q

TE58: 928-945) Semaphorin 5A (NP003857.1:

60-

78) ADAMTS-3 (O1

072: 975-989) ADAMTS-4 (CAH72146.1: 527-545) ADAMTS-19 (Q

TE59: 1096-1110) ADAMTS-10 (Q9H324: 528-545) ADAMTS-14 (CAI13857.1:980-984) Papilin (NP775733.2: 342-359) ADAMTS-13 (AAQ68465.1: 751-785)BAI-3 (CAI21873.1: 3

2-370) TSRC1 (AAH27478.1: 267-283) THSD1 (AAQ88516.1: 347-365)ADAMTS-like 2 (AAH50544.1: 54-72) ADAMTS-7 (AAH61631.1: 1576-1582) UNC5B(NP891

50.1: 595-613) ADAMTS-20 (CAD56159.3: 1478-1484) ADAMTS-5 (NP008968.1:576-568) ADAMTS-1 (Q9LH-28: 566-584) THSD6 (AAH40520.1: 44-60) CILP(AAQ88263.1: 156-175) C6 (AAB69433.1: 30-48) VSGP/F-spondin (BAB18461.1:567-5

3) CILP-2 (AAN17826.1: 153-171) ADAMTS-8 (Q9UP79: 534-552) CTGF(CAC44023.1: 204-220) ADAMTS-6 (NP922932.2:

47-

63) CYR61 (AAR05668.1: 234-250) UNC5

 (AAQ88514.1: 269-277) WISP-3 (CAB16556.1: 1

1-207) ADAMTS-9 (Q8P2N4: 1335-1351) WISP-2 (AAQ89274.1: 199-215)ADAMTS-10 (CAC82612.1: 987-113) Tum2 α1(CIV) (CAH74130.1: α2(CIV)(CAH71368.1: 1517-1593) α4(CIV) (CAA56943.1: 1499-1575) 1479-1558) α

6(CIV) (CAI40758.1: 1501-1577) α5(CIV) (AAC27816.1: 1495-1572) Tum3α5(CIV) (AAC27816.1: α6(CIV) (CAI40758.1: 1518-1

3

) α4(CIV) (CAA58943.1: 1514-1633) 1510-1529) α2(CIV) (CAH71356.1: 1

32-1

51) α1(CIV) (CAH74130.1: 1494-1513) Tum4 α5(CIV) (AAC27816.1: α2(CIV)(CAH71366.1: 1648-1684) ADAM-12 (O43184: 6

2-674) 162

-1644) α6(CIV) (CAI40758.1: 1630-1648) ADAM-9 (Q13443: 649-661) α1(CIV)(CAH74130.1: α4(CIV) (CAA56943.1: 1628-164

) 1610-1628) Tum7 α1(CIV) (CAH74130.1: α

(CIV) (CAI40756.1: 1525-1545) 1504-1523) α2(CIV) (CAH71385.1: 1542-1561)α5(CIV) (AAC27816.1: α4(CIV) (CAA58943.1: 1524-1543) 1520-1539) Angio-Macrophage stim. 1 (AAH48330.1: 388-448) IPA (AAO34406.1: 213-296)statin K1 Thrombin/cf II (AAL77438.1: 106-188) ROR-1 (CAH71706.1:312-392) HGF (P14210: 127-208) ROR-2 (Q01974: 315-394) Lp(a)(NP005568.1: 4123-4201) KREMEN-1 (BAB40958.1: 31-115) Hyahuronan binding(NP004123.1: 192-276) Hageman factor XII (AAM97832.1: 215-296) Angio-HGF (P14210: 304-363) AK-38 protein (AAK74167.1: 11-93) statin K2Macrophage stim. 1 (AAH46330.1: 188-268) ROR-1 (CAH71706.1: 310-391)Lp(a) (NP005566.1: 3560-3839) Thrombin/cf II (AAL77436.1: 105-186) ROR-2(Q01974: 314-394) tPA (AAO34406.1: 214-296) KREMEN-2 (BAD7142.1: 35-119)Angio- Lp(a) (NP005568.1: HGF (P14210: 304-377) AK-38 protein(AAK74187.1: 13-80) statin K3 1615-1690) Macrophage stim. 1 (AAH48330.1:370-448) ROR-2 (Q01974: 314-391) ROR-1 (CAH71708.1: 311-388) Thrombin/cfII (AAL77438.1: 107-183) Hagerman factor XII (AAM97932.1: 216-292)KREMEN-1 (BAB40969.1: 31-114) tPA (AAO3448.1.1: 214-293) Angio- Lp(a)(NP005568.1: HGF (P14210: 304-383) ROR-1 (CAH71706.1: 311-381) statin K44225-4508) AK-38 protein (AAK74187.1: 12-94) Thrombin/cf II (AAL77456.1:107-186) Macrophage stim. 1 (AAH48330.1: 368-449) KREMEN-1 (BAB40959.1:31-114) ROR-2 (Q01974: 314-395) tPA (AAO34406.1: 213-297) Hagermanfct/cf XII (AAM97932.1: 214-295) Hyahuronan binding (NP004128.1:192-277) Angio- Lp(a) (NP005568.1: Macrophage stim. 1 (AAH48330.1:370-448) HGF (P14210: 127-207) statin K5 1615-1690) tPA (AAM52248.1:5-89) AK-3

 protein (AAK74187.1: Thrombin/cf II (AAL77486.1: 105-18

) 14-93) ROR-2 (Q01874: 315-395) ROR-1 (CAH71706.1: 313-591) KREMEN-1(BAB40968: 51-114) KREMEN-2 (BAD97142.1: 34-118) Hagerman fct/cf XII(AAM97932.1: 214-295) Gro-β/ GRO-γ/MIP-2β/CXCL

 (AAA83184.1: THBG-β/CXCL7 (AAB46677.1: 64-121) GCP-2/CXCL5 (AAH13744.1:51-107) CXCL2 43-100) ENA-78/CXCL5 (AAP35483.1: 51-107) MIG/CXCL9(Q07325: 32-91) GRO-α/CXCL1 (AAP35526.1: PF-4/CXCL4 (AAK29643.1: 43-100)44-101) IL-8/CXCL

 (AAP35730.1: 35-94) GH-1 GH-2 (CAG45700.1: 28-180) Placental lactogen(AAP35572.1: 26-180) Somatolberin (g

225905: 26-145) IP-10/ GCP-2/CXCL

 (AAH13744.1: 47-108) CXCL10 Kininogen Hemopexin (P02790: 233-248)Lactogen GH-1 (NP00050

.2: Somatolberin (gi225905: 26-145) 26-1

0) GH-2 (CAG45700.1: 26-160) MIG/ Gro-β/CXCL2 (AAH15753.1: 42-97) CXCL9GRO-γ/MIP-2β/CXCL3(AAA

3184.1: 41-9

) ENA-78/CXCL5 (AAP35454.1: 48-103) GRO-α/CXCL1 (AAP35528.1: 42-87)THBG-β/CXCL7 (AAB46577.1: 62-117) IP-10/CXCL10 (AAH10954.1: 29-86)GCP-2/CXCL8 (AAH13744.1: 48-103)

indicates data missing or illegible when filedTable 3 shows identified similarity hits with respect to the proteins orprotein fragments with known anti-angiogenic activity. Similarities withthe anti-angiogenic proteins or protein fragments of 20-60 amino acids(PF-4, TIMP-2/loop6, TSPs, and Tums) and of 100-150 amino acids(angiostatin kringles, CXCs, somatotropins and kininogen) wereidentified in a BLAST search against the human proteome. The resultswere filtered and similarities, based on a scaled score, of 80-100% areidentified as “identical,” 60-80% as “highly similar,” and 45-60% as“similar.” The queries (first column) are listed alphabetically. Thehits are displayed with their identification number and thecorresponding part of their sequence that is identical. They areorganized according to the degree of similarity with the query.

In order to identify the conserved domains present within a querysequence, the NCBI's conserved domain (CD) search option (Marchler-Bauerand Bryant, Nucleic Acids Res, 32:W327-31, 2004) was used as part of theprotein BLAST algorithm. The EMBL's SMART (Letunic et al., Nucleic AcidsRes, 32:D142-4, 2004) sequence analysis was then used to identifyconserved domains. Information for each hit, including localizationwithin the protein as well as the protein's biological function, wascollected using the EMBL's Harvester database (Liebel et al.,Bioinformatics, 20:1962-3, 2004). In the case of proteins with knownactive domains, the total query protein sequence was used and not justthe 20-amino acid fragment. Subsequently, the active domains werelocalized within the query and classified as to whether they belonged toor included specific conserved domains.

For sequence similarity searches, the p-BLAST algorithm was used. Eachsearch was performed for human proteins using the default options of thealgorithm, except that the expectation value was increased from 10 to1000 and the word size was decreased from 3 to 2. These modificationswere made because the small length of some queries (15-20 amino acids)suggested that results calculated using these low-level criteria mightbe significant and therefore should not be rejected ab initio. Proteinswith significant similarities were initially identified using theselow-level criteria. For each of the queries, approximately 1000 initialhits having sequence similarity were identified. The results includedhigh-similarity hits, such as the maternal protein from which theanti-angiogenic fragment originated, as well as intermediate andlow-level similarity results.

A Monte Carlo-type algorithm was used to filter the initial hits oncethe e-values of the results as provided by the NCBI's p-BLAST wereconsidered. The proteins with e-values smaller than 0.1 where collected.Given that these hits were found using the p-BLAST search, there wassome evidence for similarity between the query and the hit. It wastherefore reasonable to expect that the score for the alignment of atruly similar p-BLAST hit with the initial query would be higher thanthe scores obtained by aligning a random sequence of amino acids withthis same p-BLAST hit.

By conserving the amino acid composition of the initial query, i.e. theoriginal anti-angiogenic protein, and after randomly permuting the aminoacid locations, a set of 100 new random sequences for each of thequeries was created. These random sequences were later used to determinea cut off value designating the random vs. non random identities. Thecut-off value for a random alignment was determined as follows. A set of100 random sequences was created for each of the queries by randomlyaltering an amino acid position within each sequence. The similarity ofeach of these 100 artificial sequences to each of the initial p-BLASThits was calculated and the resulting scores were fitted to an extremevalue distribution probability density function. This distributiondescribes the probability that the alignment score is a random hit,rather than a significant hit. The scores for the original protein'slocal alignment with each of the p-BLAST hits was then superimposed onthe scores of each of the random sequences, and those having scoresgreater than the random sequence cut-off value were retained. Thepercentage identity score of the random sequences was less than 20%. Thesequences with score greater than 50% only were retained. As significantidentity, a score greater than 80% was considered.

Local alignments were performed with the Smith-Waterman algorithm usingthe BLOSUM 50 substitution matrix as the scoring matrix. The BLOSUM 50matrix identifies highly similar proteins that are likely to sharesimilar overall structure whether they have distant evolutionary originsor are closely related sequences (Pearson, Methods Mol Biol,132:185-219, 2000). The resulting score for each alignment, in bits, wasscaled using as a scaling factor the score of the alignment of theinitial query with itself. As a threshold for similarity a scaled scoreabove 40% was used. All the algorithms were implemented in Matlab(Mathworks, Natick, Mass.).

The results in Table 3 are presented in the three sections correspondingto the classifications shown in Table 1: (1) anti-angiogenic proteinswith a conserved domain and known active fragment; (2) anti-angiogenicproteins with a conserved domain and unknown active fragment; and (3)anti-angiogenic proteins without conserved domains. The top 202identified similarity hits for the proteins or protein fragments withknown anti-angiogenic activity are displayed in Table 3. The firstcolumn includes the query sequence.

Anti-Angiogenic Proteins with a Conserved Domain and Known ActiveFragment

Based on the characteristic domains contained within the anti-angiogenicfragments or containing these fragments, eight protein families withestablished anti-angiogenic effects were identified. These proteinfamilies are: the type 1 thrombospondin containing proteins, the C—X—Cchemokines, the collagens, the somatotropins, the serpins, the LamininGlobular (LG) domain containing proteins, the kringle containingproteins and the complement component 1 (C1q) containing proteins.Others that do not contain a characteristic domain, such as the tissueinhibitors of metalloproteinases (TIMPs) were also identified. Whileeach of these proteins may contain multiple conserved domains, theproteins are categorized here by the conserved domain that is mostlikely to have anti-angiogenic activity based on its proximity to aknown anti-angiogenic sequence. Once the proteins were categorized bycharacteristic domains, they were compared to all the known humanprotein sequences that contain the same conserved domains, and theresults were clustered based on similarity criteria.

Proteome Query

The identification of similarities was also extended beyond the proteinswith similar conserved domains to all of the proteome. Thus, theconserved domains identified in the first column of Table 3, were usedto query all sequences present in the human proteome; the results werefiltered in order to identify truly similar hits. The result of thisprocedure was the identification of a set of peptides that may exertanti-angiogenic effects, based on similarity to the known activefragments of the anti-angiogenic proteins listed in Table 1. This set ofidentified anti-angiogenic proteins/peptides is displayed in Table 3.The function of these proteins, including their anti-angiogenicproperties, is described below.

Anti-angiogenic Peptides Related to Thrombospondins

Thrombospondins (TSPs) 1 and 2 are matricellular proteins with thewell-characterized ability to inhibit angiogenesis in vivo and toinhibit the migration and proliferation of cultured microvascularendothelial cells in vitro (Carpizo and Iruela-Arispe, Cancer MetastasisRev, 19:159-65, 2000; de Fraipont et al., Trends Mol Med, 7:401-7,2001). Angiogenesis in developing tumors and in various models of woundhealing is diminished or delayed by the presence of thrombospondin 1 or2 (de Fraipont et al., Trends Mol Med, 7:401-7, 2001). Both of theseproteins have similar structural organizations and contain three copiesof a similar conserved domain, the type I TSP domain. These conserveddomains are designated TSP1.1, TSP1.2 and TSP1.3, starting at the aminoterminus, according to their localization within the thrombospondinsequence. Other proteins with known anti-angiogenic properties and typeI TSP domains, include the Brain-specific Angiogenesis Inhibitors(BAI-1, BAI-2 and BAI-3) and the ADAMTS-1 and 8 (METH-1 and METH-2).BAI-1, a known potent angiogenesis inhibitor, which contains five TSP1domains while BAI-2 and BAI-3 have four. ADAMTS-1/METH-1 contains threeTSP1 domains and ADAMTS-8/METH-2 two.

BAI-1 is a p53 inducible gene (Nishimori et al., Oncogene, 15:2145-50,1997), that encodes a transmembrane protein that is primarily expressedin normal but not cancerous brain tissue. BAI-1 is proteolyticallycleaved at a conserved domain, the G-protein coupled receptorproteolytic cleavage site (GPS), releasing a 120 kDa extracellularfragment, vasculostatin (Kaur et al., Oncogene, 24:3632-42, 2005).Vasculostatin contains all five TSP1 domains and exhibits potentanti-angiogenic activity (Koh et al., Exp Cell Res, 294:172-84, 2004).

ADAMTS-1/METH-1 and ADAMTS-8/METH-2 belong to the metallospondin familyof proteins, a growing family of matrix metalloproteinases (MMPs). Thesetwo proteins show similarities to the reprolysin subfamily, whichincludes the ADAM proteins. METH-1 and METH-2 display anti-angiogenicproperties and inhibit endothelial cell proliferation (Iruela-Arispe etal., Ann N Y Acad Sci, 995:183-90, 2003; Luque et al., J Biol Chem,278:23656-65, 2003; Rodriguez-Manzaneque et al., J Biol Chem,275:33471-9, 2000; Vazquez et al., J Biol Chem, 274:23349-57, 1999). Thecatalytic activity of METH-1 has been experimentally shown to berequired for its anti-angiogenic effects, although TSP1 repeats aloneare able to suppress VEGF-induced angiogenesis in vitro (Luque et al., JBiol Chem, 278:23656-65, 2003). These results indicate that there are atleast two potential mechanisms to account for the METH-1 activity. It ishypothesized that the catalytic domain is required for the cleavage andrelease of the TSP1 domains, either directly in an autocatalytic manneror indirectly after binding to another METH-1 and cleaving its activefragment (Iruela-Arispe et al., Ann N Y Acad Sci, 995:183-90, 2003).

Another of the known anti-angiogenic proteins with TSP1 domains,thrombospondin 1, has been extensively mapped and its activity has beenlocalized (Tolsma et al., J Cell Biol, 122:497-511, 1993). Severalpeptides derived from thrombospondin 1 have been shown to possessanti-angiogenic properties. The pro-collagen domain of the protein hasbeen shown to exert anti-migrational effects, whereas the two activedomains MAL-II and MAL-III, residing within the TSP1.2 and TSP1.3conserved domains, respectively, are anti-angiogenic (Tolsma et al., JCell Biol, 122:497-511, 1993).

Given that the TSP1 conserved domain is the main locus ofanti-angiogenic activity in TSP1-containing proteins, the similaritybetween all the known TSP1 domains in the proteome and thrombospondin's1 TSP1.2 and TSP1.3 was queried. These two domains, as well as thecorresponding active fragments MAL-II and MAL-III, exhibit highsimilarity to the TSP1 conserved domains of hemicentin, semaphorin 5Aand 5B, and a plethora of ADAMTS enzymes including ADAMTS-2, 4, 6, 7,10, 12, 13, 14, 16, and 20 (FIG. 1). Similarities were also identifiedwithin papilin, CILP-2 and the netrin receptors UNC-5B, 5C, and 5D. TheTSP1.1 domain of thrombospondin 1 shares no similarity with any of theseproteins (FIG. 1).

TSP1.1 of the ADAMTS Proteins

The high similarity of the TSP1.1 of the ADAMTS proteins with MAL-II andMAL-III indicates that this specific domain is likely to act as a potentanti-angiogenic regulator. The first type I TSP motif is not clusteredwith the other domains, but is positioned upstream from the other TSP1domains, which are usually clustered downstream. This uniquelocalization presumably makes it more accessible to cleavage.Furthermore, in most of the ADAMTS molecules the TSP1.1 domain islocated near a cysteine-rich, also conserved, domain that functionallyregulates the binding affinity of the molecule.

Hemicentin (Fibulin 6), CILP-1 and 2, Papilin, and UNCs

The above-described strategy also identified the following proteins:hemicentin (fibulin 6), CILP-1 and 2, papilin, and UNCs, which are ofparticular interest. Hemicentin a membrane-bound cell adhesion moleculewith 48 tandem Ig modules. Hemicentin is expressed in fibroblasts,retinal pigment epithelial cells, and endothelial cells. It affects themechanical stabilization of the germline syncytium, the anchorage ofmechanosensitive neurons to the epidermis and the organization ofhemidesmosomes. Cartilage Intermediate Layer Protein 1 and 2 (CILP-1 andCILP-2) are primarily expressed in articular chondrocytes and aresecreted proteins that affect the cartilage scaffolding; papilin is aproteoglycan sulfated protein. UNCs are transmembrane netrin receptorsthat direct axon extension and migration during neural development.Because of their high similarity to the anti-angiogenic fragments ofthrombospondin 1, fragments of these proteins represent potentialangiogenesis inhibitors.

TSP1.2 of BAI-2 and BAI-3 and TSP1.3 of BAI-1

Although BAIs have been shown to exhibit anti-angiogenic activity, thisactivity has not been previously localized within the molecule. Thesequence similarities of each of the BAI's TSP1 domains to the analogousdomains of MAL-II and MAL-III and the clustering of similarities of BAIswith the predicted hits obtained using the MAL sequences (FIG. 1)suggest that the second TSP1 domain (TSP1.2) of BAI-2 and BAI-3 and thethird TSP1 domain of BAI-1 is likely to have anti-angiogenic activity.TSP1.2 shows diffuse similarity with most of the predicted proteins(FIG. 1). The BAI-2 TSP1.2 domains are also highly similar tothrombospondin's 1 TSP1.2 while the BAI-3 TSP1.2 domain is similar tothrombospondin's 1 TSP1.2 and TSP1.3. For BAI-1, it is the thirdthrombospondin domain (TSP1.3) that exhibits similarity.

WISP-1, F-Spondin, Properdin, C6, NOVH and CYR61

Finally, a BLAST search of the active domains MAL-II and MAL-III againstthe whole proteome, identified a number of hits that included proteinswith TSP conserved domains (Table 2). These hits include WISP-1,F-spondin, properdin, the complement component C6, the neuroblastomaexpressed-NOVH and CYR61 (Table 3). WISP-1, NOVH, and CYR61 belong tothe connective tissue growth factor family and have been previouslyidentified as having pro-angiogenic activity. Based on their similarityto the anti-angiogenic MAL-II and MAL-III, the results reported hereinindicate that proteolytic processing of these proteins is likely toyield angiostatic fragments.

CXC Proteins

The second cluster of proteins belongs to the CXC protein family andcontains the CXC conserved domain. These proteins include the PlateletFactor 4 (PF-4/CXC ligand 4), the Monochine Induced by Gamma interferon(MIG/CXC ligand 9), the Interferon gamma induced Protein 10 (IP-10/CXCligand 10), the Interferon inducible T-cell Alpha Chemo-attractant(ITAC/CXC ligand 11) and the B-Cell Attracting Chemokine 1 (BCA-1/CXCligand 13) (Belperio et al., J Leukoc Biol, 68:1-8, 2000; Romagnani etal., Trends Immunol, 25:201-9, 2004; Strieter et al., Semin Cancer Biol,14:195-200, 2004).

CXC chemokines are heparin-binding proteins that contain four conservedcysteines, with the first two separated by a non-conserved residue (i.e.C—X—C). The N-terminal portion of most CXC chemokines contains threeamino acid residues, Glu-Leu-Arg (ELR); this ELR motif precedes thefirst cysteine of the primary structure of these cytokines. It has beenspeculated that CXC cytokines that contain the ELR motif (ELR⁺) arepotent promoters of angiogenesis (Romagnani et al., Trends Immunol,25:201-9, 2004); in contrast, those lacking the ELR motif (ELR⁻) arepotent inhibitors of angiogenesis (Romagnani et al., Trends Immunol,25:201-9, 2004). However, it has also been shown that in Lewis lungcarcinoma cells, an ELR⁺ protein, the beta GRO protein (CXC ligand 2) isan angiogenesis inhibitor (Cao et al., J Exp Med, 182:2069-77, 1995).

The most well-studied CXC angiostatic protein is PF-4 (Bikfalvi, SeminThromb Hemost, 30:379-85, 2004; Bikfalvi and Gimenez-Gallego, SeminThromb Hemost, 30:137-44, 2004). The ability of PF-4 to bind heparin andheparan sulfate glycosaminoglycans (GAGs) with high affinity appears tobe important for several of its biological functions. PF-4 inhibitsendothelial cell migration, proliferation, and in vivo angiogenesis inresponse to bFGF and VEGF by binding to their receptors or by formingPF4-VEGF and PF4-bFGF heterodimers that impair the binding of growthfactors to their receptors (Bikfalvi, Semin Thromb Hemost, 30:379-85,2004). There are several known isoforms of PF-4; the numbering hererefers to the originally reported sequence (Deuel et al., Proc Natl AcadSci USA, 74:2256-8, 1977). The PF-4¹⁷⁻⁷⁰ fragment is the naturallyoccurring fragment produced by proteolytic processing of full lengthPF-4 by elastase. The 1-17 amino acid domain remains attached to thecleaved protein through a disulfide bond. PF-4⁴⁷⁻⁷⁰/CTF, the C-terminalfragment of full-length PF-4, is a very potent angiostatic fragment(Bikfalvi and Gimenez-Gallego, Semin Thromb Hemost, 30:137-44, 2004).This fragment is contained within the CXC conserved domain.

The active fragment of other CXC family members has not been previouslyidentified. Hypothesizing that a peptide fragment having anti-angiogenicactivity resides within their CXC conserved domains, the similarities ofeach of these CXC domains with the corresponding domains of putativeanti-angiogenic proteins was determined. The CXC domain of GRO-β,although an ELR protein, is considered to be anti-angiogenic as well.FIG. 2A illustrates the notably high similarity between the CXC domainsof known angiostatic proteins and those of ELR⁺ proteins such as GRO-α,GRO-β, ENA-78 and GCP-2. This similarity suggests that even though thefull-length proteins may be angiogenic, fragments within their CXCdomains are angiostatic

PF-4's anti-angiogenic activity was localized to a specific portion ofthe CXC domain and the sequence of this active fragment was used tosearch for similar protein fragments. These similarities were identifiedfirst for the pool of all CXC domains and then for the total humanproteome. Surprisingly, the active domain of PF-4 was found to sharesimilarity with fragments of the CXC domains of GRO-α, GRO-β, GCP-2,ENA-78, and THBG-2 (FIG. 2A), which are ELR⁺ proteins, providing supportfor the proposition that fragments of these proteins may beanti-angiogenic.

A BLAST search of the active domain of PF-4 against the whole proteomewas performed and the results of this search were filtered using theMonte Carlo algorithm. These results are summarized in Table 3.Excluding the CXC-related proteins, the hits include the CC motifchemokines 1, 2, and 19 and lymphotactin, all of which exhibitedrelatively low similarities but were still above the similaritythreshold. Finally, the CXC domains of the chemokines with knownanti-angiogenic activities, but unknown active fragments are alsosearched against the proteome and the results are filtered. The resultsindicated that the CXC domain of BCA-1 shares similarities with proteinsother than CXC, such as the CC motif ligands 3, 7, and 13. Thecorresponding domains of the other members of the CXC family showed nosignificant similarities.

Collagen Type IV-Derived Fragments

A significant source of anti-angiogenic fragments is thenon-collagenous/NC1 domain of various type IV-collagen a fibrils. Thereare six type IV-collagen α fibrils; α1-α6. Each is composed of acysteine-rich 7S domain at the N-terminal of the protein, which isinvolved in the covalent assembly of four type IV collagen fibers into aplanar macrostructure; a central triple-helical collagenous domain; anda C-terminal NC1 domain, that is involved in the self-assembly of αchains into heterotrimers. The α1 and α2 chains are the predominantlyexpressed forms in most of the tissues; the α3-α6 are found inspecialized basement membranes.

Fragments of α1(CIV) NC1 (arresten) (Colorado et al., Cancer Res,60:2520-6, 2000), α2(CIV) NC1 (canstatin) (Kamphaus et al., J Biol Chem,275:1209-15, 2000), α3(CIV) NC1 (tumstatin) (Maeshima et al., J BiolChem, 276:15240-8, 2001) have been shown to inhibit angiogenesis andtumor cell proliferation, although the corresponding domains of the restof the α(CIV) fibrils have not yet been shown to exhibit similarproperties. From the aforementioned domains, the anti-angiogenicproperty of tumstatin has been localized to four specific fragments ofα3(CIV) NC1: Tum2, Tum3, Tum4, and Tum7 (Maeshima et al., J Biol Chem,275:21340-8, 2000; Maeshima et al., J Biol Chem, 276:31959-68, 2001).

The NC1 domains of all the type IV collagen a fibrils comprise twocopies of the conserved C-terminal tandem repeated in the type IVprocollagen domain, C4.1 and C4.2. Surprisingly, the active domains oftumstatin can be localized within and do not span both of the conserveddomains. Each of them independently belongs to either C4.1 or C4.2.Moreover, the most potent fragments, Tum2, Tum3, and Tum7, are localizedonly to C4.1 while the remaining Tum4 to C4.2.

A bioinformatics approach was used to test the similarities of thevarious C4 domains of all the type IV collagen α fibrils to the activefragments of tumstatin. Similarity criteria were used to predict (FIG.2B) that the C4.1 domains of α5 and α6 fibrils might exhibitanti-angiogenic effects. It is likely that their angiostatic potentialresides within the specific fragment of the NC1 domain and not the totalsequence. Furthermore, each of the C4.1 and C4.2 domains was found tobear similarity only to the corresponding domains of the collagenfibrils (FIG. 2B).

When tumstatin's active domains were compared with the whole proteome,only Tum4 produced significant hits. It shares similarity with ADAM-9and ADAM-12 and to a lower degree with tenascin C. ADAM-9 and -12 aremembrane-anchored proteins implicated in a variety of biologicalprocesses involving cell-cell and cell-matrix interactions, includingfertilization, muscle development, and neurogenesis. For both of theseproteins, the similarity regions are localized to the extracellularportion of the molecule. Tenascin C is a substrate adhesion moleculethat appears to inhibit cell migration and is a ligand for most of theintegrins. From its sequence similarity to tumstatin it also appearsthat a fragment of the molecule may be anti-angiogenic.

Anti-Angiogenic Proteins with a Conserved Domain and Unknown ActiveFragment

In all of the aforementioned protein families, at least one member has aknown active domain, and the anti-angiogenic activity was localizedwithin an approximately 20-amino acid domain. This information was usedto predict the function of similar domains in related family members. Inaddition, using information regarding the site of the active domain,similarities were identified with other members of the protein family aswell as with more distantly related proteins that possessed this domain.

Below, protein families are classified by their conserved domains. Theseproteins have been shown to exhibit anti-angiogenic effects, but theiractive domains have not yet been localized. It is likely that theiractive domains are hidden within the sequence of their common conserveddomains and similarities were identified based on this hypothesis. Thefact that these queries have thus far identified anti-angiogenicactivity within these conserved domains supports this hypothesis.

Serpins

Serine proteinase inhibitors (serpins) modulate the activity of serineproteinases that function in coagulation, fibrinolysis, complementactivation, and phagocytosis (van Gent et al., Int J Biochem Cell Biol,35:1536-47, 2003). Members of this family with potent angiostaticactivity include Pigment Epithelium Derived Factor (PEDF), maspin,antithrombin, kallistatin and a fragment of angiotensinogen.

The anti-angiogenic activity of PEDF selectively effects newly formingvessels, while sparing existing vessels (Bouck, Trends Mol Med, 8:330-4,2002). In vivo, PEDF blocks angiogenesis that has been induced in theretina by ischemia or by growth factors in the cornea. In both cases,PEDF causes endothelial cells involved in forming new vessels toinitiate apoptosis (Abe et al., Am J Pathol, 164:1225-32, 2004). PEDF'sserpin domain is unique; it does not share significant similarity withany other serpin domain and shares only minor similarity with α2antiplasmin's serpin (FIG. 2C). Both of the proteins belong to the sameclade, clade F of the serpins. α2 antiplasmin is a serine proteaseinhibitor that inhibits plasmin and trypsin and inactivateschymotrypsin. Recently, two functional anti-angiogenic protein siteswere identified in PEDF: a 34 amino acid sequence and a TGA domain of 10amino acids were shown to be responsible for the anti-angiogenicproperties of the protein (Filleur et al., Cancer Res, 65:5144-52,2005). These sites extend beyond the serpin domain. The 34 amino acidsequence and the TGA domain sequence were used to directly search forsimilarities with other proteins within the proteome. Interestingly,this search yielded no similarity hits. PEDF is unique at both the levelof its serpin domain and of its anti-angiogenic protein sites.

Maspin (mammary serpin) is a member of the serpin super family and anovel protease inhibitor related to other inhibitors such as plasminogenactivator inhibitor and α1-antitrypsin, as well as to non-inhibitorserine proteins such as ovalbumin (Maass et al., Acta Oncol, 39:931-4,2000). In vitro invasion assays have demonstrated that endogenous andexogenous maspin inhibits invasion of endothelial cells through thebasement membrane matrix (Maass et al., Acta Oncol, 39:931-4, 2000;Schaefer and Zhang, Curr Mol Med, 3:653-8, 2003). Maspin belongs toglade B of the serpins, and is similar in its conserved domain toleukocyte elastase inhibitor, squamous cell carcinoma antigen 1,cytoplasmic antiproteinases 2 and 3, and placental thrombin inhibitor(FIG. 2C). Interestingly, these proteins are all cytoplasmic. Thesquamous cell carcinoma antigen is secreted into plasma by tumor cells.These proteins exhibit serine-type endopeptidase inhibitor activity.Leukocyte elastase inhibitor regulates the activity of the neutrophilproteases elastase, cathepsin G and proteinase 3. Placental thrombininhibitor inhibits thrombin while the cytoplasmic antiproteinase 3inhibits granzyme B. They have no known anti-angiogenic function, butspecific fragments of the proteins may have such a function.

Antithrombin is a plasma-derived, single-chain glycoprotein. A complexmolecule with multiple biologically important properties, it is a potentanticoagulant and also has anti-inflammatory properties, several ofwhich are related to its participation in the coagulation cascade. Boththe reactive loop of antithrombin resulting from cleavage by thrombinand its latent form exhibit anti-angiogenic activity (O'Reilly et al.,Science, 285:1926-8, 1999). Antithrombin belongs to Glade C of serpins.Its conserved domain is similar to the corresponding serpin domain ofcytoplasmic proteinase inhibitor 2 and placental thrombin inhibitor(FIG. 2C).

Kallistatin is found in vascular smooth muscle cells and in endothelialcells of human blood vessels. It has multiple biological functionsincluding blood pressure regulation, protection against inflammation,vasculature relaxation, and stimulation of neointimal hyperplasia.Kallistatin also possesses anti-angiogenic properties (Miao et al.,Blood, 100:3245-52, 2002; Miao et al., Am J Physiol Cell Physiol,284:C1604-13, 2003). Wild-type kallistatin, but not a mutant formlacking the heparin binding activity that resides within the conservedserpin domain, inhibits VEGF-induced proliferation, growth, andmigration of human microvascular endothelial cells (Miao et al., Am JPhysiol Cell Physiol, 284:C1604-13, 2003). Kallistatin belongs to cladeA and its conserved serpin domain is similar to the correspondingdomains of α1-antichymotrypsin, protein C inhibitor/PAI-3,corticosteroid binding globulin, thyroxine binding globulin and germinalcenter B-cell expressed protein (FIG. 2C). All of these hits areextracellular serine-type endopeptidase inhibitors, and function as partof the coagulation cascade. α1-antichymotrypsin inhibits neutrophilcathepsin G and cell chymase while both protein C inhibitor and germinalcenter B-cell expressed protein inhibit protein C and plasminogenactivators. Corticosteroid and thyroxin binding globulins are majorhormone transport proteins, circulating in the blood of mostvertebrates.

Angiotensinogen is the precursor of angiotensin I, an inactivedecapeptide that is converted into angiotensin II, the main effector ofthe renin-angiotensin system. Full length angiotensinogen (AGT¹⁻⁴⁵²),des[Ang I] angiotensinogen (AGT¹¹⁻⁴⁵²), and RCL-cleaved angiotensinogen(AGT¹⁻⁴¹²), a C-terminal cleaved product of the protein areanti-angiogenic (Celerier et al., Hypertension, 39:224-8, 2002). Searchfor similarities between angiotensinogen's serpin domains and otherserpin motifs did not produce any significant results (FIG. 2C).Similarly, no significant similarities were found to proteins from thewhole proteome.

Somatotropin Hormones

The somatotropins share a conserved domain but the activeanti-angiogenic fragment has not been previously identified. This familyincludes growth hormone, lactogen, and prolactin, which are proteinswith known angiostatic effects.

Prolactin (PRL) is a polypeptide hormone synthesized and secretedprimarily by the lactotroph cells of the anterior pituitary. It is alsosynthesized at extrapituitary sites including the mammary epithelium,placenta, uterus, brain, and immune system (Harris et al., Ann Med,36:414-25, 2004). PRL participates in the regulation of reproduction,osmoregulation, and immunomodulation. Growth hormone (GH) is involved inregulating growth and morphogenesis. Growth hormone is produced in largepart by the anterior pituitary in all vertebrates. The human placentaexpresses two structural homologs of hGH, human placental lactogen (hPL)and a variant of hGH (hGH-V). Members of the PRL/GH family and derivedpeptides have been reported to both stimulate and inhibit angiogenesis(Struman et al., Proc Natl Acad Sci USA, 96:1246-51, 1999). In general,the full length members of the human PRL/GH family, i.e., hPRL, hPL, andhGH stimulate vessel formation whereas their cleaved 16-kDa N-terminalfragments are anti-angiogenic both in vivo and in vitro. Proteolyticcleavage of PRL occurs at sites of PRL production via cathepsin D,resulting in two fragments of 16 kDa (16K-hPRL) and 6 kDa. It can alsobe cleaved by kallikerin producing a 22 kDa fragment. It is the 16K-hPRL(N-terminal fragment) that possesses anti-angiogenic properties andinhibits prostate tumor growth in mice by suppressing blood vesselformation. Cleaved PRL has been observed in mouse, rat, and human serum,whereas free 14- and 16-kDa PRL appear to be secreted by thehypothalamoneurohypophyseal system of the rat. Human GH is cleaved byplasmin, thrombin, and subtilisin, yielding similar fragments (Strumanet al., Proc Natl Acad Sci USA, 96:1246-51, 1999).

These three anti-angiogenic proteins, the 16-kDa fragments of growthhormone, placental lactogen and prolactin contain the somatotropinhormone conserved domain in their sequences. Similarity identificationwas directed towards proteins that contained the somatotropin hormoneconserved domain. The identified proteins are somatotropin hormonefamily isoforms of the initial set of three queries. In all of the casesthe somatotropin conserved domain or a large part of it is containedwithin the 16-kDa fragments, already known to have anti-angiogenicproperties.

Interestingly, the somatotropin domain of prolactin exhibits nosimilarity with any of the domains of the same family; it seems to be aunique moiety (FIG. 2D). In contrast, both GH and PRL share significantsimilarities with the somatotropin domains of most of the other familymembers. Specifically, the somatotropin domains of both pituitary andplacental specific growth hormone are highly similar to each other andalso share similarities with the corresponding domains of lactogen andthe lactogen (chorionic somatomammotropin) CS-5. The conserved domain oflactogen is similar to the somatotropin domains of the two growthhormone isoforms and lactogen's CS-5 (FIG. 2D).

The most interesting result arose from a direct search for similaritiesof the 16-kDa anti-angiogenic fragments within the total proteome.Again, prolactin's 16-kDa fragment showed no similarity with any otherprotein; surprisingly, however, the 16-kDa fragments of growth hormoneand lactogen were found to be highly similar to somatoliberin (growthhormone releasing factor). Somatoliberin belongs to the glucagon proteinfamily and is secreted from the hypothalamus as a pre-protein that iscleaved to a 44-amino acid fragment that stimulates growth hormonerelease from the pituitary. As noted above, the full-lengthsomatotropins are pro-angiogenic; somatoliberin is considered to be anangiogenesis stimulator. The high similarity of somatoliberin to the16-kDa anti-angiogenic fragments indicates that this protein may exhibitanti-angiogenic behavior.

Each of these protein families with shared conserved domains and unknownactive fragments comprises multiple protein members. Four of the knownanti-angiogenic proteins contain a characteristic conserved domainwithin their sequences, although only single members of thecorresponding protein families have been identified as angiostatic.These proteins are perlecan's fragment endorepellin, which contains thelaminin globular conserved domain and angiostatin which contains thekringle domain, both discussed in the following sections.

Laminin Globular Domain-Containing Proteins

Perlecan is a major basement membrane heparan sulfate proteoglycan. Itis involved in the stabilization of other basement membrane moleculesand regulates cell adhesion as well as vessel permeability. TheC-terminal of perlecan, endorepellin, has been shown to inhibitendothelial cell migration, tube formation in collagen and angiogenesisin CAM and matrigel assays (Bix and Iozzo, Trends Cell Biol, 15:52-60,2005; Mongiat et al., J Biol Chem, 278:4238-49, 2003). Endorepellinconsists of three LamG conserved domains LamG1 (LG1), LamG2 (LG2), andLamG3 (LG3), which are separated by two EGF-like domains within eachpair of LamG modules. Physiologically endorepellin is cleavedproteolytically by MMP-1 and MMP-3 releasing either the three-LamGdomain cassette or the last LamG domain, LamG3.

The similarity of the globular laminin domains of endorepellin to othersequences of the same family is insignificant. The second and thirddomains, LamG2 and LamG3, both displayed similarity only with the LamG1and LamG2 domains of another proteoglycans, agrin. Agrin is anotherbasement membrane protein that induces the aggregation of acetylcholinereceptors and other post-synaptic proteins in muscle fibers and it iscrucial for the formation of the neuromuscular junctions. The structuralsimilarity of the C-terminal of agrin to endorepellin, as manifested bythree consecutive LamG domains, separated by two EGF-like motifs,suggests that the C-terminal region may also possess anti-angiogenicactivity.

A direct search for similarities between the LamG domains of perlecanand the whole proteome did not provide any additional significantresults.

Kringle Containing Proteins

One of the most thoroughly studied angiogenesis inhibitor is angiostatin(O'Reilly et al., Cell, 79:315-28, 1994). The structure of angiostatinincludes the first four kringle (K) domains of plasminogen. Kringlestructures exist in many proteins, and such proteins can containanywhere from one to several kringles. The primary amino acid sequenceof each kringle domain consists of approximately 80 amino acids. Kringleproteins do not share a common function, but act as growth factors,proteases, or coagulation factors. Although most kringle-containingproteins have only one kringle domain, the glycoprotein, apolipoprotein(a), is unusual in containing 38 kringle domains. Plasminogen, theparent protein of angiostatin, contains a total of five kringle domainsand the fifth domain is excluded from angiostatin. Like other individualkringle domains, the K5 domain of human plasminogen contains 80 aminoacid residues. Primary structure alignment shows that K5 exhibitsremarkable sequence identity (˜60%) with K1, although K2, K3, and K4also share significant homologies with K5. Both proteolytic/denaturedand appropriately folded recombinant K5 displays more potentpro-apoptotic effects on endothelial cells than do the other individualkringle domains. In fact, K5 alone exhibits a several-fold greatereffect than do the K1-4 of angiostatin. This unexpected finding suggeststhat K5 might inhibit endothelial cell growth via a separate mechanism,or that K5 is a more potent inducer of inhibitory targets on endothelialcells. It has been shown in various in vitro studies that the rankingorder for kringle potency in inducing endothelial cell apoptosis isK5>K1·K2·K3 complex>K1·K2·K4 complex>K1>K3>K2>K4 (Cao and Xue, SeminThromb Hemost, 30:83-93, 2004).

The bioinformatics approach described herein has proven useful inidentifying fragments similar to angiostatin's kringles. The most potentangiogenesis inhibitor, Kringle 5, was found to share similarities withthe following kringle-containing proteins: Macrophage stimulating factor1, hepatocyte growth factor, lipoprotein/Lp(a), receptor tyrosine kinaselike orphan receptor 1 and 2, coagulation factor II (thrombin) and XII(Hageman factor), hyaluronan binding protein 2, tissue plasminogenactivator and the kringle containing proteins KREMEN-1 and 2.

Kringles 1-4 are similar to a smaller group of proteins includingmacrophage stimulating factor 1, hepatocyte growth factor andlipoprotein/Lp(a). All of the aforementioned proteins contain multiplekringle domains within their sequences and plasminogen exhibitssimilarity with most of them. In particular, K5 is extremely similar tothe last 38^(th) kringle of apolipoprotein localized next to atrypsin-like serine protease domain as well as to the anti-angiogenicprotein AK-38, a protein that consists only of the last kringle ofapolipoprotein (FIG. 2E). Lipoprotein which consists of 38 kringles(kringles 2 to 29 are identical) demonstrates different modes ofsimilarity with plasminogen's kringles. All of its kringles, apart fromthe 38^(th), are notably similar to plasminogen's K3 and K4, whereasonly the last kringle is similar to plasminogen's K5. The clustering ofthe lipoprotein's similarities around different plasminogen's kringlessuggests that K5 is a separate moiety, distinct from kringles 1-4.

Another candidate is macrophage stimulating factor 1, which belongs tothe family of granulocyte/macrophage colony-stimulating factors. Theseare cytokines that contribute to hematopoiesis by controlling theproduction, differentiation, and function of the white cells of theblood including the granulocytes and the monocytes/macrophages. It playsa role in immunological defense, bone metabolism, lipoproteinsclearance, fertility, and pregnancy. The four kringles of macrophagestimulating factor principally display similarities to plasminogen's K1,K2 and K3 (FIG. 2E).

Hepatocyte growth factor (HGF) is a potent mitogen for matureparenchymal hepatocytes. It also seems to be a hepatotrophic factor andacts as growth factor for a broad spectrum of tissues and cell types.HGF contains four kringles; among them kringles 2 and 3, which exhibitsignificant similarities to plasminogen's K2, 3, and 4 (FIG. 2E). It istherefore likely that they have potent anti-angiogenic activity. Otherproteins with significant similarities to plasminogen's kringle 5 arereceptor tyrosine kinase-like orphan receptors 1 and 2, thrombin,Hageman factor (cf XII), hyaluronan binding protein 2, and tissueplasminogen activator (Table 2). All of these proteins may possessanti-angiogenic properties in addition to their known role as mediatorsof the blood coagulation cascade.

Complement Component C1q Containing Proteins

Type VIII collagen has been demonstrated to contain fragments withpotent anti-angiogenic. This protein is an ECM component that issynthesized by endothelial cells, keratinocytes, mast cells and tumorcells. It is a hetero-trimer composed of two α1 and one α2 fibrils. TheNC1 domain of the α1(CVIII) fibril displays anti-angiogenic activitylike that of endostatin (Xu et al., Biochem Biophys Res Commun,289:264-8, 2001). However, the activity has not yet been mapped to aspecific site on the protein.

Interestingly, the α1(CVIII) NC1 domain contains a conserved domain, thecomplement component C1q domain, that spans most of the fragments andpossibly differentiates it functionally from endostatin. Thebioinformatics approach described above was used to investigate thesimilarity between the C1q conserved domain contained within theangiostatic fragment of type VIII collagen and other C1q domains. Thisdomain is highly similar to the corresponding domains of the α2 fibrilof collagen VIII and the α1 fibril of collagen X. Thus, both of thesefragments may possess angiostatic properties similar to those ofα1(CVIII) NC1. A direct search for similarities between the C1q domainof the α1(C VIII) NC1 and of the full-length NC1 fragment and the wholeproteome also identified this same group of proteins.

Anti-Angiogenic Proteins Without Conserved Domains

Finally, there is a very small group of known angiogenesis inhibitorsfor which the angiostatic activity is precisely mapped to specificactive domains within the sequence of the protein. The proteins do notcontain any previously identified conserved domains within theirsequences. Most of the angiostatic active domains are approximately20-60 amino acids long (Table 1) including kininogen, loop 6 of tissueinhibitor of metalloproteinases 2, calreticulin and the collagen XVIIIand XV endostatins.

High-molecular-weight kininogen (HK) is a plasma protein with a varietyof physiological functions. Originally identified as a precursor ofbradykinin, a bioactive peptide that regulates many cardiovascularprocesses, HK is now known to play important roles in fibrinolysis,thrombosis, and inflammation. HK binds to endothelial cells where it canbe cleaved by plasma kallikrein to release bradykinin (BK). Theremaining portion of the molecule, cleaved HK, is designated cleavedhigh-molecular-weight kininogen or HKa (Guo and Colman, J ThrombHaemost, 3:670-6, 2005). While BK has been intensively studied, thephysiological implications of HKa generation are not clear. Recentstudies have revealed that HKa inhibits angiogenesis while BK promotesit (Guo and Colman, J Thromb Haemost, 3:670-6, 2005). Mapping studieshave indicated that a peptide (HK⁴⁸⁶⁻⁵⁰²) in the D5 region of HKinhibits cell migration, while the peptide HK⁴⁴⁰⁻⁴⁵⁵ is responsible forinhibiting cell proliferation (Colman et al., Blood, 95:543-50, 2000;Guo et al., Arterioscler Thromb Vasc Biol, 21:1427-33, 2001). Our searchfor similarities between the HK⁴⁴⁰⁻⁴⁵⁵ active domain and other proteinfragments within the proteome identified hemopexin, which binds heme andtransports it to the liver for breakdown and iron recovery, as well asthe relatively less similar isoforms of the solute carrier family 39protein, which is an integral membrane protein with zinc transportproperties.

Moses and colleagues (Fernandez et al., J Biol Chem, 278:40989-95, 2003)have demonstrated the uncoupling of the MMP-inhibitory andanti-angiogenic activities of TIMP-2 using the Pichia pastorisexpression system to engineer and produce both the N- and C-terminaldomains of TIMP-2. They found that although both domains inhibitedangiogenesis in the embryonic CAM assay, the C-terminal domain andwild-type TIMP-2 were more effective inhibitors of angiogenesis in themouse corneal pocket assay than was the N-terminal TIMP-2 domain.Furthermore, the inhibitory ability of the N-terminal domain wasdependent on MMP-inhibitory activity. The ability of TIMP-2 to inhibitendothelial cell proliferation was localized to the C-terminal domain ofTIMP-2, and specifically to the C-terminal disulfide loop, loop 6. Loop6 showed significant similarities to the corresponding domains of TIMP-4and TIMP-3 and, to a lower degree to, TIMP-1.

Calreticulin is a unique endoplasmic reticulum (ER) luminal-residentprotein. This protein affects many cellular functions, both within theER lumen and outside the ER environment. In the ER lumen, calreticulinperforms two major functions: chaperoning and regulating Ca²⁺homeostasis. Vasostatin, the N-terminal domain of calreticulin inclusiveof amino acids 1-180, is an angiogenesis inhibitor that shows antitumoreffects in vivo (Pike et al., J Exp Med, 188:2349-56, 1998; Pike et al.,Blood, 94:2461-8, 1999). Like vasostatin, the whole protein,calreticulin, selectively inhibits endothelial cell proliferation andangiogenesis, but not cells of other lineages. Calreticulin lacking itsN-terminal 1-120 amino acids inhibits endothelial cell proliferationthat is comparable to that of vasostatin. The internal fragment 120-180of calreticulin inhibits angiogenesis and is probably the proteinfragment responsible for the angiostatic effects of vasostatin.Vasostatin showed no similarity to any other protein except for a distalhit, calnexin, which is also an ER calcium-binding protein.

A very significant source of a potent anti-angiogenic fragment,endostatin, is type XVIII collagen (O'Reilly et al., Cell 88; 277-85,1997). The 20- to 22-kDa fragment of NC1 of the α chain of CXVIII wasidentified as endostatin. A similar fragment of the NC1 domain of the α1chain of CXV has been shown to have properties similar to those ofCXVIII-endostatin (Sasaki, et al., J Mol Biol 301;1179-90, 2000;Ramchandran et al., Biochem Biophys Res Commun 255; 735-9, 1999). The22-kDa fragment of NC10 of the α1 chain of CXV was identified as restin,which shows anti-angiogenic effects similar to those of endostatin.These two fragments apart from their similarity to one another did notshow any other significantly similar hits in a BLAST search andfiltering against the whole proteome.

The present invention provides a systematic means of classifying knownendogenous anti-angiogenic proteins and protein fragments. Based onsimilarity criteria a number of similar sequences were identified. Thesesimilarities suggest that the polypeptides and fragments identifiedexhibit anti-angiogenic properties. In the case of most of the proteinsidentified using the bioinformatic analysis, the relationships to knownanti-angiogenic proteins and fragments have not been previouslyidentified. These newly identified proteins and fragments constitutenovel drug candidates for anti-angiogenesis therapeutics.

To date, the identification of potentially anti-angiogenic proteins hasbeen performed purely empirically. Selected proteins wereproteolytically processed by proteases and the cleaved fragments testedin various in vitro and in vivo angiogenesis protocols. This process hasyielded a number of proteins and considerable information regarding thelocalization of the anti-angiogenic protein site, i.e., the proteinregion responsible for a particular effect. In some cases a specificprotein site of 20-60 amino acids length has been identified; however,in most cases the anti-angiogenic behavior has been attributed to longerfragments of 100-150 amino acids length or even to the whole protein.The identification of protein sites and the observation that these sitesare localized within the considered conserved domains suggests that theanti-angiogenic behavior is localized within this conserved domain.Identification of similarities between the conserved domains of multipleproteins belonging to the same protein family and the anti-angiogenicfragments has yielded novel candidates that may exhibit similarfunctions. This conclusion was based solely on the amino acid sequencesof the hits and not on structural criteria. Moreover, in the case ofproteins for which the active anti-angiogenic site has not yet beenmapped, similarity criteria has been used to identify putative regionsof their amino acid sequence in which angiostatic activity is localized.

Based on the bioinformatic analysis of the whole human proteome, thepool of anti-angiogenic candidates consists of extracellularmatrix-residing or circulating proteins. In some cases the novel hitsare fragments of proteins with known pro-angiogenic activity. As shownfor the somatotropin hormones, it is likely that while the whole proteinpromotes angiogenesis, small fragments of the protein areanti-angiogenic. This property defines a novel category of proteins withdual behavior. Without wishing to be bound by theory, it is likely theexpression of enzymes that processes the initial protein into smallerfragments that controls the function of such proteins.

It is also possible that the shift from the pro-angiogenic to theanti-angiogenic function is controlled by a small subset of amino acids.For proteins identified herein, the proteins with known pro-angiogenicbehavior that show similarity to known anti-angiogenic fragments arehighly similar to these known fragments but not identical to them.Again, without wishing to be bound by theory, it is possible that thesubset of amino acids that differ between the two proteins controlstheir angiogenic properties. Potential mutations within these subsetscould provide a control mechanism for the transition between the twototally different properties.

This analysis is the first computational approach that attempts toidentify novel endogenous angiogenesis inhibitors. The knowledge ofspecific protein sites at which the anti-angiogenic behavior residesprovides for the identification of novel candidates. Thesecomputationally identified candidates are then tested experimentally foranti-angiogenic properties as described herein. This process of miningangiogenesis inhibitors requires combined computational and experimentalefforts. Such an integrative approach results in a systematic andefficient identification of anti-angiogenic protein fragments as well asadvancing current understanding of the control of the angiogenic switch.

Example 2 Results of Proliferation Experiments

As described above, computational bioinformatic algorithms were used toidentify a set of protein fragments of naturally occurring, endogenousproteins that may possess potent anti-angiogenic properties. Thealgorithms identified approximately 200 similarity hits for the proteinsor protein fragments with known anti-angiogenic activity. Of the 200hits, some of which are duplicated, over 150 represent distinct novelputative anti-angiogenic protein fragments. Among the novel fragments,there were hits similar to platelet factor-4, with 7 identified similarprotein domains; various fragments of thrombospondin 1, each of whichhaving 45 identified similar fragments; and various tumstatins, withapproximately 5 similar fragments each. The proteins with identifiedsimilarities and lengths of 100-150 amino acids included the angiostatinkringles; the CXC chemokines Gro-β, IP-10, and MIG, each withapproximately 8 top similar protein sites; growth hormone-1 andplacental lactogen with 3 similar fragments each; as well as kininogen,with a single identified similarity.

An experimental medium-throughput platform was used to test theidentified polypeptides and peptides biological activity. Following theNCI (National Cancer Institute) guidelines for testing theanti-angiogenic efficacy of novel agents(http://dtp.nci.nih.gov/aa-resources/aa_index.html), an endothelial cellproliferation assay was used. Endothelial cell proliferation isimportant for new capillary sprout formation; thus, inhibitingproliferation should be sufficient for a complete or partial inhibitionof angiogenesis. Where the active domains of proteins have not beenidentified, fragments that exhibit similarities to known anti-angiogenicfragments or proteins are assayed to identify amino acid sequenceshaving anti-angiogenic activity. Peptides not exhibiting activity in theproliferation assays described below, will be tested for angiogenesismodulating activity in functional assays described herein. In additionto proliferation assay, examples of a migration assay are presentedbelow as well as a Directed In Vivo Angiogenesis Assay (DIVAA).

Peptide Synthesis and Handling

The peptides are produced using the custom peptide synthesis facility inthe Department of Oncology, Johns Hopkins University, and a commercialproviders (Abgent, San Diego, Calif.; New England Peptide, Inc.,Gardner, Mass.) using a solid phase synthesis technique. For eachpeptide HPLC and mass spectroscopy analysis is performed and proof ofidentity and purity is provided. The procedure yields 10 mg of >95% purepeptide. 10 mg are aliquoted, by the manufacturer, in single mg vialsand shipped. Each single mg of peptide was diluted in 100 μl finalsolution, which provides a 10,000 μg/ml peptide concentration. The 100μl is aliquoted in 10×10 μl stock solutions and frozen.

In order to predict the proper solvent that is required to solubilizethe peptide, the hydrophobicity of the aminoacid chains is calculated.The more hydrophobic peptides require polar solvents, such as DMSO.Otherwise the peptides are diluted in a commercially available balancedsalt solution, Hanks balanced salt solution (HBSS). Where the peptide isneither strongly hydrophobic nor highly hydrophilic then the solvent isempirically determined by testing the peptides solubility in smallvolumes of solvents starting with 20 μl of HBSS. Where a peptide is notsoluble in HBSS a small solid pellet is formed at the bottom of thetube. This separation of phases is a sufficient indication that HBSS isnot the proper solvent. We then use another 80 μl of DMSO and repeat theprocess. If the peptide is soluble in the 20 μl of HBSS we add another80 μl to final volume 100 μl. So far in all of the cases this procedurehas proven satisfactory in establishing the solubility of the solidpeptides in different solvents.

For all solvents, 9×10 μl of stock solutions are stored in −80° C. andthe tenth 10 μl is used to provide a working solution. In the singlevial containing 10 μl of 10,000 μg/ml of peptide 90 μl of HBSS was addedto generate 100 μl of 1,000 μg/ml solution. Those 100 μl were aliquotedin 10×10 μl and stored at −80° C.

Experimental Protocol

The effect of the various peptides on cell proliferation was assayed, byadministering the peptides to human umbilical vein endothelial cells(HUVECs) in culture. 45 peptides belonging to four distinct families ofproteins including the thrombospondin-1 containing proteins, the C—X—Cchemokines, collagen derived peptides and peptides from tissueinhibitors of metalloproteinases were tested.

The potency of each of the peptides was tested using the proliferationassay at 2 days and 4 days following administration of the peptides anda similar assay using a different cell density at 3 days. In thefour-day experiments, the cell medium was changed after the second dayof incubation and the peptide was replaced at the initial concentrationtogether with the medium. TNP-470 is an antiangiogenic compound that wasused as a positive control for assessing anti-angiogenic efficacy. Theactivity of each polypeptide was, therefore, expressed as a percentageof TNP-470 activity, i.e., the optical density obtained experimentallyis scaled such that the negative control is 0% and the positive controlis 100% as explained below (FIG. 4). Each plate included a well havingHUVEC cells that received 0.1 μg/ml TNP-470. For example, a peptidehaving the same effect on endothelial cell proliferation would have 100%of the activity of TNP-470. Note that activity could exceed 100%.

The observed activity of the tested peptides in many of the casesreached 40% of the TNP-470 activity, and in some cases reached 80%. Athigher peptide concentrations the activities of some of the peptides arelikely to increase further. Also some peptides have biphasic activity. Apeptide having “biphasic activity” has maximum activity at anintermediate concentration, but this anti-angiogenic activity wasdiminished at very high or very low concentrations. This biphasicactivity maybe time-dependent. Peptides that show biphasic behavior attwo days may change this characteristic after four days with aconcomitant gain of activity. In most cases, peptides gain activity overtime, or maintain a constant level of activity.

Statistical significance was tested using one way and two way ANOVA;p<0.001 is indicated by **, p<0.05 by *.

Analysis of the Proliferation Experimental Results

In this assay, the effects of the predicted anti-angiogenic fragments onthe ability of HUVECs to proliferate was characterized. A decrease inthe proliferation rate of the cells indicates that those agents arecapable of disrupting the angiogenic process. A commonly usedmethodology to test for proliferation is to monitor the viability andmetabolic activity of endothelial cells, in the presence of theanti-angiogenic fragments at different concentrations and various timesteps. The cell proliferation reagent WST-1 was used as asubstrate/assay to measure the metabolic activity of viable cells. WST-1is a colorimetric, non-radioactive assay and can be performed in amicroplate. It is suitable for measuring cell proliferation, cellviability or cytotoxicity. The assay is based on the reduction a redtetrazolium salt, WST-1, by viable, metabolically active, cells to formthe yellow formazan crystals soluble in the cell medium. In contrast toother formazan based assays, such as MTT, the formazan crystals do notneed to be solubilized by the addition of a detergent; they are solvablein the cell medium.

The cells are cultured described above. Once cells have reached 80%confluence they are trypsinized and resuspended in EGM-2. The celldensity is estimated using a hemocytometer. All of the endothelial cellsused were from passage 4 to passage 8.

The proliferation assay involved two steps. During the first step thecells, approximately 5,000 cells per well for a 96-well plate, wereseeded without any extracellular matrix substrate on the wells overnight(8 hours). The initial cell culture medium was removed using amultichannel pipetor and the candidate peptides were applied. Alogarithmic scale of four different concentrations of 0.01 μg/ml, 0.1μg/ml, 1 μg/ml and 10 μg/ml of each peptide was used. Each of theconcentrations was tested in quadruplicate. The viability of the cellswas challenged at 2 and 4 days upon the application of the peptide. As apositive control for decreased viability, 0.1 μg/ml of TNP-470(0-(chloro-acetyl-carbamoyl) fumagillol, a synthetic analogue offumagillin; TNP-470 was provided by NCI, was provided along with themedium, and as a negative control for normal viability, the cells werecultured without the addition of any agent. The cells were thenincubated with 10 μl per well of the red WST-1 reagent for approximately2 to 4 hours. During this incubation period, viable cells converted, intheir mitochondria, the red WST-1 to the yellow formazan crystals whichdissolve in the medium. The second step of the assay involved thequantification of the changes in proliferation which was performed bymeasuring the change in color after lysing the cells. The samples wereread using the ELISA plate reader Victor3 V (Perkin Elmer) at awavelength of 570 nm. The amount of color produced is directlyproportional to the number of viable cells. For each plate, this valuewas compared to background by measuring the signal in a cell-free wellcontaining only cell culture medium and dye. This background signal wassubtracted from the signal from each cell-containing well. The resultingsignal was expressed as optical density (O.D.). A typical example of anoptical density output from a measurement of a single peptide that wasapplied to cells in culture for two days is shown in FIG. 3.

In this figure the optical density was plotted for each of 4wells/sample for each concentration of administered peptide. The valuewas expressed as the average of the four measurements along with thestandard deviation. The first point, which is denoted as 0 peptideconcentration, represented the background signal present in the negativecontrol cell-free well. The last point, denoted as TNP-470, representsthe collections of wells where 0.1 μg/ml of TNP-470 was applied. Thiswell served as a positive control.

The optical density results are scaled using the positive and negativecontrols. Each of the optical density measurements, from each well, wasdivided by the average optical density of the negative control (onlyfull medium) and the results were scaled from 0% to 100%. A scalingwhere 100% represents the activity of TNP-470 and 0% represents zeroanti-proliferative activity as measured in the negative control wellswas used. Using this scaling method FIG. 3 was used to generate ameasure of peptide activity as shown in FIG. 4. Note that the 2-day and4-day results were scaled by their own negative and positive controlvalues.

In addition to the 2 and 4 day proliferation experiment where 5,000cells per well were seeded, the effects of the peptides on theproliferating ability of HUVECs were also studied using cells seeded ata density of 2,000 cells per well. This lower cell seeding densityallowed the experiment to be extend for an additional day withoutchanging the cell medium. The effects of the peptides when applied tothe cells for 3 consecutive days is also reported herein. Theproliferation assay again involved two steps. During the first step2,000 cells per well for a 96-well plate were seeded without anyextracellular matrix substrate on the wells overnight (8 hours). Theinitial cell culture medium was removed using a multichannel pipetor. Tosynchronize the cells, the cells were serum starved for 4 hours and thecandidate peptides were applied. During serum starvation the cellculture medium was changed from a “full” medium to the “basal” medium,which contained no growth factors or serum. Serum starvation for 4 hourshad no effect on the proliferation of the cells. For this assay, thelogarithmic scale of four different concentrations of 0.01 μg/ml, 0.1μg/ml, 1 μg/ml and 10 μg/ml of peptide was used. In addition, a largerconcentration of 30 μg/ml peptide was also applied. Each of theconcentrations was again tested in quadruplicate.

After the third day of peptide application the WST-1 dye was applied forapproximately 2 to 4 hours (usually after 3 hours the color saturates).The proliferation was later tested on the ELISA plate reader asdescribed for the 2 and 4 day assay. Note that the 2- and 4-day and the3-day protocols differed in that 5,000 cells/well versus 2,000cells/well seeded, respectively.

Peptides from four families of proteins, two of the families containinga conserved domain (the TSP-1 containing proteins and the C—X—Cchemokines), and two families without a conserved domain (the collagensand the TIMPs) were experimentally screened in order to determine theanti-angiogenic activity of the computationally predicted fragments.

Peptides Derived from Thrombospondin-1 Containing Proteins

Thirty two peptides that contain a thrombospondin-1 domain (TSP-1) weretested in proliferation assays as described above. The results of thesetests follow.

THSD-1

Thrombospondin, type I, domain containing 1 (THSD-1) is a cell surfaceprotein that contains a domain similar to thrombospondin-1. Thepredicted anti-angiogenic fragment (QPWSQCSATCGDGVRERRR) (SEQ ID NO: 64)showed approximately 20% activity. This activity was fairly constantthroughout the 4 days of the peptide application as shown in FIG. 5A forall the applied concentrations. THSD-1 maximum activity was attained ata low peptide concentration (0.01 μg/ml). Similar activity was observedin the 3-day assay (FIG. 5B). There was a substantial increase inactivity at the higher concentration of 30 μg/ml.

THSD-3

A second isoform of the thrombospondin, type I, domain containingproteins (THSD) that contains a domain predicted to possessanti-angiogenic activity is THSD-3. The THSD-3 fragment(SPWSPCSGNCSTGKQQRTR) (SEQ ID NO: 65) did not effect endothelial cellproliferation in the two, three, or four day assay (FIGS. 6A and 6B).

THSD-6

A third isoform of the thrombospondin, type I, domain containingproteins (THSD) that contains a domain predicted to exertanti-angiogenic activity is THSD-6. The THSD-6 fragment(WTRCSSSCGRGVSVRSR) (SEQ ID NO: 66) attained a maximum activity ofapproximately 20% at 2 days and 30% at 3 days. This activity wasbiphasic in the two, three, and four day assay (FIGS. 7A and 7B). THSD-6showed maximum potency when administered at 0.1 μg/ml.

CILP

CILP is the cartilage intermediate layer protein. It contains a TSP-1domain and the predicted anti-angiogenic fragment (SPWSKCSAACGQTGVQTRTR)(SEQ ID NO: 39). CILP showed biphasic activity that was consistentlymaintained throughout the four days that the cells were incubated withpeptide application (FIG. 8A). The maximum activity of the peptide(˜30%) is observed at 1 μg/ml concentration. At lower concentrations(0.01 and 0.1 μg/ml) the activity falls to 15% and at higherconcentrations (10 μg/ml) the activity is also diminished (5%). For the3 day assay (FIG. 8B) a similar biphasic response was observed. In the3-day assay, maximum activity was observed at 0.1 μg/ml peptideconcentration.

WISP-1

WISP-1 is the WNT1 inducible signaling pathway protein 1. It is a cellsecreted protein that belongs to the connective tissue growth factor(CTGF) family. It is expressed at a high level in fibroblast cells, andoverexpressed in colon tumors. It attenuates p53-mediated apoptosis inresponse to DNA damage through activation of the Akt kinase. Thepredicted anti-angiogenic fragment (SPWSPCSTSCGLGVSTR1) (SEQ ID NO: 74)shows intermediate activity (˜20%) after four days of applicationwhereas after two days its activity is not statistically significant(FIG. 9A). The WISP-1 activity for the 3 day assay retained intermediatelevels but reached higher values at 30 μg/ml (FIG. 9D).

WISP-2

Another isoform of the WNT1 inducible signaling pathway protein 1 isWISP-2. The fragment that is derived from WISP-2, TAWGPCSTTCGLGMATRV(SEQ ID NO: 75), was quite potent at small concentrations of 0.01 μg/mland shows a biphasic response after the second day of the peptideapplication (FIG. 9B). For the 3 day assay the WISP-2 peptide retainedintermediate activity of 20% (FIG. 9E).

WISP-3

A third isoform of the same family of proteins that was predicted tocontain a fragment with putative anti-angiogenic effects is WISP-3.Similarly to WISP-2, the fragment of WISP-3, TKWTPCSRTCGMGISNRV (SEQ IDNO: 76), shows potency at small peptide concentration of 0.01 μg/mlwhich was also biphasic (FIG. 9C). During the three day applicationassay, the peptide activity increased significantly as the concentrationincreased (FIG. 9F)

F-Spondin

F-spondin, or vascular smooth muscle cell growth-promoting factor,contains a thrombospondin domain that is predicted to beanti-angiogenic. Two fragments of F-spondin were identified as havingputative anti-angiogenic activity. For the first fragment of theprotein, SEWSDCSVTCGKGMRTRQR (SEQ ID NO: 73), the activity of thepeptide after two days of application was not statistically significant.After four days there was a notable activity which was predominant atlow peptide concentration (0.01 μg/ml) and shifts the activity of thepeptide to a biphasic profile (FIG. 10A). Interestingly at a highpeptide concentration, during the 3 day assay, the peptide showsincreased activity (FIG. 10C). The second predicted fragment ofF-spondin WDECSATCGMGMKKRHR (SEQ ID NO: 72), shows a small activityafter four days of application that reached a maximum of 10% (FIG. 10B).Its activity during the two-day assay was not statistically significant(FIGS. 10C) but it exhibited activity in the 3-day assay (FIG. 10D).

CTGF

CTGF, connective tissue growth factor, is the prototype for a family ofproteins that promotes proliferation and differentiation ofchondrocytes. It also mediates heparin- and divalent cation-dependentcell adhesion in many cell types including fibroblasts, myofibroblasts,endothelial and epithelial cells. The peptide that is predicted to beanti-angiogenic (TEWSACSKTCGMGISTRV) (SEQ ID NO: 41) attained 40%activity after four days of application (FIG. 11A) and the activityappeared to saturate at an intermediate concentration (1 μg/ml). Thepeptide's potency after two days was low (FIG. 11A). CTGF showedincreased activity over time. The results of the 3-day assay resembledthose observed in the 2-day assay (FIG. 11B).

Fibulin-6

Fibulin-3 and fibulin-5 proteins have been shown to be anti-angiogenic.These fibulins belong to the EGF superfamily. Fibulin-6, orhemicentin-1, which belongs to the immunoglobulin superfamily, containsmultiple TSP-1 domains that are predicted to have anti-angiogenicactivity. Two distinct domains of fibulin-6 were identified by thebioinformatic analysis as having putative anti-angiogenic properties.One of these (ASWSACSVSCGGGARQRTR) (SEQ ID NO: 45) showed activity ofapproximately 30% in the four-day assay (FIG. 12A). Interestingly, inthe two-day assay the peptide showed biphasic activity. It attainedmaximum activity at a low concentration (0.1 μg/ml), whereas at lower orhigher concentrations it was less active. During the 3-day assay thepeptide showed similar activity to that observed after 2 days (i.e., abiphasic response that reached a 20% maximum at 0.1 μg/ml peptideconcentration (FIG. 12D). The second fragment derived from fibulin-6(QPWGTCSESCGKGTQTRAR) (SEQ ID NO: 44) showed even greater potency. Itattained maximum activity (˜40%) four days after application (FIG. 12B).The second fragment also exhibited biphasic behavior after the four daysof application. Its maximum activity was observed at the lowest testedconcentration of 0.01 μg/ml. Interestingly, in the 3-day assay,anti-angiogenic activity increased with increasing peptide concentration(FIG. 12E). The third fragment of fibulin-6 (SAWRACSVTCGKGIQKRSR) (SEQID NO: 43) was characterized by activity that reached 30% (FIG. 12C). Asimilar response was observed in the 3 day assay (FIG. 12F) with peptideactivity increasing as concentration increased.

CYR61

CYR61, or cysteine-rich angiogenic inducer 61, is a secreted,cysteine-rich, heparin-binding protein encoded by a growthfactor-inducible immediate-early gene. It is an extracellularmatrix-associated signaling molecule. CYR61 promotes the adhesion ofendothelial cells through interaction with integrin and augments growthfactor-induced DNA synthesis. The predicted fragment(TSWSQCSKTCGTGISTRV) (SEQ ID NO: 42) showed biphasic anti-proliferativeactivity on the endothelial cells. Maximum (30%) activity was observedat 0.01 μg/ml (FIG. 13A). In the 3-day assay (FIG. 13B), the activity ofthe peptide was similar, attaining maximum activity of 30% at 0.1 μg/mlconcentration.

NOVH

NOVH, or the nephroblastoma overexpressed protein, is an immediate-earlyprotein that plays a role in tumor cell growth regulation. The TSP-1domain of NOVH was predicted to exert anti-angiogenic activity. Thepredicted anti-angiogenic fragment (TEWTACSKSCGMGFSTRV) (SEQ ID NO: 46)reached 20% maximum activity (FIG. 14A) at small peptide concentrations(0.01 μg/ml) and the activity was retained throughout the four days ofthe peptide application. In the 3-day assay the activity showed amonotonic increase with concentration (FIG. 14B).

UNC-5C and UNC-5D

UNC-5C and UNC-5D belong to the UNC-5 family of netrin receptors.Netrins are secreted proteins that direct axon extension and cellmigration during neural development. They are bifunctional proteins thatact as attractants for some cell types and as repellents for others, andthese opposite actions are thought to be mediated by two classes ofreceptors. The UNC-5 family of receptors mediate the repellent responseto netrin; they are transmembrane proteins containing 2 immunoglobulin(Ig)-like domains and 2 type I thrombospondin domains (TSP-1) in theextracellular region. Fragments of the TSP-1 domains are predicted toexert anti-angiogenic activity. The fragment derived from UNC5-C(TEWSVCNSRCGRGYQKRTR) (SEQ ID NO: 70) has negligible activity at the twodays of the peptide application whereas it reached 20% activity afterfour days (FIG. 15A). For the 3 day application experiment the peptideresponse was similar to the 4 days of application with a 20% maximum at10 μg/ml. At 30 μg/ml the peptide activity increases to 40% (FIG. 15C).The fragment from UNC5-D (TEWSACNVRCGRGWQKRSR) (SEQ ID NO: 71) hasconstant activity throughout the four days assay and the activityreached 20% (FIG. 15B). The activity of UNC5-D in the 3-day assay wassimilarly biphasic, but now the maximum reached 30% at 0.1 to 1 μg/ml(FIG. 15D).

SCO-Spondin

SCO-spondin is a relative of the thrombospondin family and correspondsto glycoproteins secreted by the subcommissural organ (SCO). SCO-spondinis also known to modulate neuronal aggregation. The TSP-1 peptidefragment that is predicted to be anti-angiogenic (GPWEDCSVSCGGGEQLRSR)(SEQ ID NO: 63) showed low anti-proliferative activity which reachedmaximum efficiency ˜15% at high peptide concentrations for both of the2-4 and 25% for 3-day assays (FIGS. 16A and 16B).

Properdin

Properdin or factor P, is a plasma protein that is active in thealternative complement pathway of the innate immune system. A fragmentfrom its TSP-1 domain (GPWEPCSVTCSKGTRTRRR) (SEQ ID NO: 49) waspredicted to exert anti-angiogenic activity. The peptide attainedmaximum activity 30% at the lowest tested concentration (0.01 μg/ml)(FIG. 17A). After four days of peptide application, it showed a biphasicresponse with maximum activity 20% observed at low concentrations (0.01μg/ml and 0.1 μg/ml). In the 3-day proliferation assay (FIG. 17B) theactivity significantly increased at 30 μg/ml.

C6

Another protein that functions as part of the immune system is thecomplement component 6 protein (C6). C6 is a component of complementcascade. It is part of the membrane attack complex that can insert intothe cell membrane and cause cell to lyse. The predicted anti-angiogenicfragment (TQWTSCSKTCNSGTQSRHR) (SEQ ID NO: 38), which is part of theTSP-1 domain that C6 contains, exhibited maximum activity of 35% at highpeptide concentrations after four days of incubation with cells (FIG.18A). The activity of C6 was different from the activity observed inproperdin, the other peptide fragment that belongs to the immuneresponse pathway. The proliferation results in the 3-day assay (FIG.18B) were similar to those observed in the 2 day assay. The activity wasbiphasic with a 25% maximum reached at intermediate peptideconcentrations (FIG. 18B).

ADAMTS Family

A large portion of the fragments that contain TSP-1 domains and arepredicted to exert anti-angiogenic activity belong to the ADAMTS familyof proteins. Members of the family share several distinct proteinmodules, including a propeptide region, a metalloproteinase domain, adisintegrin-like domain, and a thrombospondin type 1 (TSP-1) motif. Thepeptides tested that belong to this protein family are thrombospondinrepeat containing 1 protein (TRSC1) or ADAMTS-like-4, ADAMTS-4,ADAMTS-8, ADAMTS-16 and ADAMTS-18.

TRSC1

TSRC1 or ADAMTS-like-4, is a protein with a predicted anti-angiogenicfragment (SPWSQCSVRCGRGQRSRQVR) (SEQ ID NO: 69) that showed minimalactivity on the endothelial cell proliferation ability after two days ofthe peptide application. After four days there was prominent activity atintermediate peptide concentrations. The activity profile was biphasicand reached a maximum of 25% at 0.1 μg/ml (FIG. 19A). The experimentalresults for the 3-day assay (FIG. 19B) were similar to those in the4-day assay. The peptide activity increased at the highest appliedconcentrations of 30 μg/ml.

ADAMTS-4

The ADAMTS-4 protein contains a TSP-1 domain (GPWGDCSRTCGGGVQFSSR) (SEQID NO: 7) that is predicted to be anti-angiogenic. When tested for itsanti-proliferative activity the ADAMTS-4 fragment reached a maximum of35% efficiency at its highest tested concentration (10 μg/ml) after fourdays of the peptide application (FIG. 20A). Similar results wereobserved in the 3 day assay (FIG. 20B).

ADAMTS-8

The second protein that belongs to the ADAMTS family and contains aTSP-1 domain that is predicted to be anti-angiogenic is ADAMTS-8. Thefragment from ADAMTS-8 (GPWGECSRTCGGGVQFSHR) (SEQ ID NO: 14) exertedmaximum activity of 35% at concentration 1 μg/ml after two days of thepeptide application (FIG. 21A). A maximum activity of 30% at 1 μg/ml wasobserved in the 4-day assay also. The peptide's activity was reduced atthe maximum tested concentration of 10 μg/ml relative to its activity atlower concentrations. This biphasic response was observed in the two,three, and four day assays (FIGS. 21A and 21B).

ADAMTS-16

ADAMTS-16 is another protein that contains a TSP-1 domain that ispredicted to be anti-angiogenic. The ADAMTS-16 fragment(SPWSQCTASCGGGVQTR) (SEQ ID NO: 24) showed strong activity at four daysand exhibited a biphasic response with a maximum 30% observed at 0.01μg/ml and 0.1 μg/ml concentrations (FIG. 22A). At concentrations greaterthan 0.1 μg/ml the activity of the peptide was reduced. In the 3-dayassay, an increase in peptide activity was observed at the highestapplied concentration of 30 μg/ml (FIG. 22B).

ADAMTS-18

ADAMTS-18 is another protein containing a TSP-1 domain that wasexperimentally tested for its anti-proliferative activity. The activityof the ADAMTS-18 derived fragment (SKWSECSRTCGGGVKFQER) (SEQ ID NO: 29)was similar to that of ADAMTS-8 (FIG. 23A). The peptide exhibited 30%activity at 1 μg/ml after two days of incubation with cells (FIG. 23A).As was observed for ADAMTS-8, the peptide's activity was reduced at 10μg/ml relative to its activity at lower concentrations. This biphasicresponse was observed in the two and four day assays. In the 3-day assayat the highest peptide concentration (30 μg/ml) the peptide's activitywas significantly increased.

Semaphorins

The semaphorins are another family of proteins that contain a TSP-1domain. Two fragments of semaphorin 5A and one fragment of semaphorin 5Bwere predicted to have anti-angiogenic activity. Semaphorins areinvolved in axonal guidance during neural development and some of themhave been shown to possess anti-angiogenic activity, although Semaphorin5A and 5B have not been identified as anti-angiogenic agents.

Semaphorin 5A and 5B

The first semaphorin 5A fragment (GPWERCTAQCGGGIQARRR) (SEQ ID NO: 60)exhibited minimum anti-proliferative activity after two days of thepeptide application and showed intermediate activity in the 4-day assay.The activity reached a maximum of 25% at concentrations higher than 1μg/ml (FIG. 24A) for the 2 and 4 day assay whereas reached 35% in the 3day assay (FIG. 24D). The second semaphorin 5A fragment testedSPWTKCSATCGGGHYMRTR (SEQ ID NO: 61) showed no anti-proliferativeactivity (FIGS. 24B and 24E). The semaphorin 5B fragment,TSWSPCSASCGGGHYQRTR (SEQ ID NO: 62) showed 10% anti-proliferativeactivity (FIG. 24C). Similar results were obtained in the 3-day assay(FIGS. 24D-F).

Papilin

Another protein that belongs to the TSP-1 containing proteins ispapilin. Papilin is a proteoglycan-like sulfated glycoprotein thatfunctions during development. Two fragments of papilin contained withindistinct TSP-1 domains of the protein sequence were testedexperimentally for their ability to suppress endothelial cellproliferation. The first fragment of papilin, GPWAPCSASCGGGSQSRS (SEQ IDNO: 48), showed potent anti-proliferative activity of 30% (FIG. 25A).The second fragment of papilin, SQWSPCSRTCGGGVSFRER (SEQ ID NO: 47), wasless potent and showed only minimal anti-proliferative activity (FIG.25B). Similar results were obtained for these fragments in the 3-dayassay (FIGS. 25C and 25D).

ADAMs

Another family of proteins that contain a TSP-1 domain are the ADAMs.Members of this family are membrane-anchored proteins and have beenimplicated in a variety of biologic processes involving cell-cell andcell-matrix interactions, including fertilization, muscle development,and neurogenesis. ADAM proteins interact with SH3 domain-containingproteins, bind mitotic arrest deficient 2 beta protein, and are alsoinvolved in TPA-induced ectodomain shedding of membrane-anchoredheparin-binding EGF-like growth factor.

ADAM-9

A putative anti-angiogenic fragment was identified in ADAM-9. Theprotein fragment, KCHGHGVCNSNKN (SEQ ID NO: 1), showed ˜20%antiproliferative activity (FIG. 26A). Similar activity was observed inthe 3-day proliferation assay (FIG. 26C).

ADAM-12

A fragment derived from the ADAM-12 isoform, MQCHGRGVCNNRKN (SEQ ID NO:2), showed activity at 2 days at concentrations of 1 and 10 μg/ml (FIG.26B). Similar results were observed in the 3-day assay, with theactivity increasing as the peptide concentration increased (FIG. 26D).

Peptides Derived from C—X—C Chemokines

Six peptides derived from C—X—C chemokines were predicted to exhibitanti-angiogenic activity based on their similarity to knownanti-angiogenic protein fragments of this family.

Gro-α/CXCL1

Gro-α or CXCL1 exhibits chemotactic activity for neutrophils. It alsoplays a role in inflammation and showed its effects on endothelial cellsin an autocrine fashion. The peptide fragment (NGRKACLNPASPIVKKIIEKMLNS)(SEQ ID NO: 102) predicted to exert anti-angiogenic properties showed ananti-proliferative activity of 15%. This activity remained constant forthe four days of testing (FIG. 27A). The peptide showed maximum activityat 0.01 and 0.1 μg/ml but showed reduced activity at higherconcentrations. At the maximum tested peptide concentration, 10 μg/ml,its activity was statistically insignificant. In the 3-day proliferationassay, the peptide activity was significantly increased at the highesttested concentration of 30 μg/ml (FIG. 27B).

Gro-γ/CXCL3

Gro-γ or CXCL3 showed similar chemotactic properties to Gro-α. Thepredicted anti-angiogenic fragment, NGKKACLNPASPMVQKIIEKIL (SEQ ID NO:106), showed 15% activity after four days of incubation and exhibited aslight gain of activity during two extra days of incubation. Maximumactivity was observed at the lowest applied peptide concentration of0.01 μg/ml both at the 2 and 4 and 3 day assays (FIGS. 28A and 28B).

THBG/CXCL7

Beta thromboglobulin (THBG), or CXCL7, is a platelet-derived growthfactor that belongs to the C—X—C chemokine family. It is a potentchemoattractant and activator of neutrophils. It has been shown tostimulate various cellular processes, including DNA synthesis, mitosis,glycolysis, intracellular cAMP accumulation, prostaglandin E2 secretion,and synthesis of hyaluronic acid and sulfated glycosaminoglycan. Thepeptide, DGRKICLDPDAPRIKKIVQKKL (SEQ ID NO: 114), was predicted to haveanti-angiogenic activity. It showed 15% activity (FIG. 29A) at 10 μg/ml.Similar activity was observed in the 3-day assay (FIG. 29B).

IL-8/CXCL8

Interleukin 8 (IL-8) or CXCL8 is one of the major mediators of theinflammatory response. This chemokine is secreted by several cell types.It functions as a chemoattractant, and is also a potent angiogenicfactor. A peptide fragment derived from IL-8, DGRELCLDPKENWVQRVVEKFLK(SEQ ID NO: 110), showed 20% anti-proliferative activity at its maximumapplied concentration (10 μg/ml) at the 2 and 4 day assay (FIG. 30A).During the 3-day assay its activity is higher at 30 μg/ml (FIG. 30B).

ENA-78/CXCL5

ENA-78 or CXCL5 is an inflammatory chemokine that also belongs to theC—X—C chemokine family. This chemokine is produced concomitantly withinterleukin-8 (IL-8) in response to stimulation with eitherinterleukin-1 (IL-1) or tumor necrosis factor-alpha (TNF-α). Thischemokine is a potent chemotaxin involved in neutrophil activation. TheENA-78 derived peptide, NGKEICLDPEAPFLKKVIQKILD (SEQ ID NO: 95), waspredicted to be anti-angiogenic. It showed approximately 30% activity at2 days even at the lowest tested concentration tested (0.01 μg/ml) (FIG.31A). In the 3 day assay this activity was reproducible over all testedconcentrations (FIG. 31B). At highest concentrations of 30 μg/ml thepeptide showed high activity.

GCP-2/CXCL6

A fragment derived from a C—X—C chemokine, CXCL6, showed similaractivity in the 2-4 day and in the 3-day assay. GCP-2 or CXCL6 is achemotactic protein that contains a fragment predicted to beanti-angiogenic (NGKQVCLDPEAPFLKKVIQKILDS) (SEQ ID NO: 98) (FIGS. 32Aand 32B). The peptide derived from GCP-2 showed biphasic behavior inboth the 2-4 and in the 3-day assays. It showed 30% activity at 2 daysat 0.01 μg/ml. At higher peptide concentrations peptide activity wasreduced. Virtually no anti-proliferative activity was observed at 10μg/ml. In the 3-day assay its activity was increased at 30 μg/ml (FIG.32B).

Peptides Derived from Collagen

The anti-angiogenic activity of two peptides derived from collagensequences was tested. A peptide from the alpha 6 fibril of type 4collagen and a peptide from alpha 5 fibril of type 4 collagen.

Alpha 6 Fibril of Type 4 Collagen

The first peptide was derived from the alpha 6 fibril of type 4 collagen(C4α6). A peptide having the sequence: YCNINEVCHYARRNDKSYWL (SEQ ID NO:93) showed 15% anti-proliferative activity at four days (FIG. 33A). Asimilar profile was observed in the 3-day proliferation assay as well(FIG. 33B) though at 30 μg/ml concentration, the peptide activityincreased to 80%.

Alpha 5 Fibril of Type 4 Collagen

The second set of peptides derived from a collagen sequence that waspredicted to have anti-angiogenic activity was derived from the alpha 5fibril of type 4 collagen (C4α5). There are two such peptides. The firstpeptide, LRRFSTMPFMFCNINNVCNF (SEQ ID NO: 89), showed 80% activity atits highest applied concentration, 10 m/ml (FIG. 34A) for the 2- and4-day proliferation assay. In the 3-day assay, the peptide activity forthe common concentrations was reproducible and increased even more whentesting the higher concentration of 30 μg/ml (FIG. 34C). The secondpeptide, SAPFIECHGRGTCNYYANS (SEQ ID NO: 91), showed low activity at twoor four days though during the 3-day assay its activity increases at thehighest applied concentration of 30 μg/ml (FIGS. 34B and 34D).

Alpha 4 Fibril of Type 4 Collagen

The third set of peptides derived from collagens with putativeanti-angiogenic activity are derived from the alpha 4 fibril of type 4collagen (C4α4). The first peptide, AAPFLECQGRQGTCHFFAN (SEQ ID NO: 87),has intermediate activity following two days of incubation (FIG. 35A).The activity was similar during the 3-day assay (FIG. 35D) though theactivity increased with increasing the applied peptide concentration.The second peptide, LPVFSTLPFAYCNIHQVCHY (SEQ ID NO: 85), exhibitedsimilar behavior (FIGS. 35B and 35E). The third peptideYCNIHQVCHYAQRNDRSYWL (SEQ ID NO: 86) showed low anti-proliferativeactivity at 2 or 4 days (FIG. 35C). In the 3-day assay its activity wasgreater and was increasing at higher peptide concentrations (FIG. 35F).

Tissue Inhibitors of Metalloperoteinases (TIMP)

A peptide derived from a sequence of a tissue inhibitor ofmetalloproteinases (TIMP) was tested. The peptide, which was derivedfrom TIMP-3, ECLWTDMLSNFGYPGYQSKHYACI (SEQ ID NO: 155), was predicted tohave anti-angiogenic activity. This fragment showed a maximum 30%activity at two days (FIG. 36A). A similar response was observed duringthe 3-day assay (FIG. 36B).

Scrambled Peptides

To determine if the anti-proliferative activity was specific, the effectof scrambled peptides was examined. These peptides contained the sameamino acids as the studied peptides but the order of the amino acidswithin the peptide sequence was randomly permuted. For example, theC—X—C derived GCP-2/CXCL6 with the original sequenceNGKQVCLDPEAPFLKKVIQKILDS (SEQ ID NO: 98), which showed a biphasicresponse having 35% maximum anti-proliferative activity was permuted tothe random sequence QDVFNKDGKVILLSPQAICLPKEK (SEQ ID NO: 158). Also, theTIMP3 derived peptide, ECLWTDMLSNFGYPGYQSKHYACI (SEQ ID NO: 155), whichshowed 30% maximum anti-proliferative activity was randomly permuted toLCMTKSDCYQPAWYIHEGFYNLSG (SEQ ID NO: 159). The anti-proliferativeeffects of these randomly scrambled peptides was not statisticallysignificant at any concentration for the 2- and 4-day assays (FIGS. 37Aand 37B).

Example 3 Results of In-Vivo Screening

A directed in vivo anti-angiogenesis assay (DIVAA) was used to test theanti-angiogenic efficacy of the alpha 5 fibril of collagen type 4, whichhad the sequence: SAPFIECHGRGTCNYYANS (SEQ ID NO: 91). This peptideshowed anti-angiogenesis activity that increased as its concentrationincreased in in vitro assays. This peptide showed only intermediateactivity in in vitro screening assays. The peptide was solubilized inbuffer solution at 200 μg/ml without an organic solvent. DIVAA is areproducible and quantitative in-vivo method of assaying angiogenesis.It involves preparation of silicon cylinders of 20 μl volume, closed onone side, filled with some type of extracellular matrix (for exampleMatrigel or BME-basement membrane extract) with or without premixedangiogenic factors. These angioreactors are then implantedsubcutaneously in the dorsal flank of mice. Accompanied with the onsetof angiogenesis, vascular endothelial cells migrate into theextracellular matrix and form vessels in the angioreactor. As early asnine days post-implantation, there are enough cells to determine aneffective dose response to angiogenic modulating factors.

A set of DIVAA angioreactors was prepared. Each of them was filled witha basement membrane extract (extracellular matrix) containing thepeptides and growth factors. The basement membrane extract used was theCulturex® extract (Trevigen, Inc., Gaithersburg, Md.), which is asoluble form of basement membrane purified from Engelbreth-Holm-Swarm(EHS) tumor. The extract gels at 37° C. to form a reconstituted basementmembrane. The major components of the Basement Membrane Extract includelaminin I, collagen IV, entactin, and heparan sulfate proteoglycans. Theextract used was growth factor free, which means that the matrix doesnot contain any inherent growth factors that are applied exogenously. Asa positive control, 37.5 ng of bFGF and 12.5 ng of VEGF per 200 μl ofthe basement membrane extract in accordance with the manufacturer'sdirections (Trevigen, Inc., Gaithersburg, Md.). As a negative control abioreactor containing the reduced growth factor only basement membraneextract was used. For the peptide application, the extracellular matrixwas mixed with the positive control constituents, 37.5 ng of bFGF and12.5 ng of VEGF per 200 μl of matrix, and with 200 μg/ml of the alpha 5fibril of collagen type 4 peptide (sequence: SAPFIECHGRGTCNYYANS) (SEQID NO: 91). Two reactors per condition and per mouse were used. Eachcondition was repeated in quadruplicate. The DIVAA reactors wereimplanted subcutaneously in the abdomen of C57BL/6 female mice. Twoangioreactors were implanted per mouse, one on either side of theabdomen, 4 mice were used per condition tested.

Eleven days after implantation the mice were sacrificed and theangioreactors were removed from their abdomens. FIG. 38A showsmicrophotographs from the angioreactors for each of the conditions.Fewer endothelial cells invaded the extracellular matrix containing theinhibitory peptide than were observed in the negative control. In thepanel at the far left, the positive control angio-reactors having welldeveloped vasculature is shown. In the middle panel is the angioreactorcontaining the inhibitory peptide and at the right is the negativecontrol. The figure shows that the peptide strongly inhibitedangiogenesis.

To obtain a quantitative assessment of the angiogenic invasion, thecontent of the angioreactors was removed from the cylinder and theendothelial cells were stained using FITC-Lectin (FIG. 38A) Once thetubes were extracted from the mice, the inside of the angioreactors wasrinsed with 300 μl of a 36 kDa, bacillus-derived neutralmetalloproteinase which is commercially available as Cellsperse(Trevigen, Inc., Gaithersburg, Md.), an extracellular matrix digestingsolution and the content of the reactors as well as the rinse wastransferred into a microtube. After one hour of incubation, the tubeswere centrifuged at 250×g for 5 minutes at room temperature. The cellpellets and insoluble fractions were retained. The pellet wasresuspended in 500 μl of full cell medium (EGM-2, Clonetics) to allowfor cell surface receptor recovery, and incubated at 37° C. for onehour. The cells were then centrifuged at 250×g for 5 minutes at roomtemperature to collect cell pellets. The pellets were resuspended in aFITC-Lectin solution, and incubated at 4° C. overnight.

Fluorescence of the FITC-Lectin solution was quantitated by measuringthe fluorescence at 485 nm excitation and 510 nm emission using afluorescence plate reader (Victor 3V, Perkin Elmer). The intensity ofthe signal was directly proportional to the number of endothelial cellsthat were present in the angioreactors. The results were scaled to apercentage scale so that 100% would represent the mean of the positivecontrols (FIG. 38B).

Example 4 Analysis of Peptide Motifs

By performing multiple sequence alignment to the sequences of thepredicted peptides, the conservation of specific motifs that were commonin many of the sequences was observed. Multiple sequence alignment wereperformed using the ClustalW algorithm to the sequences of the peptidesthat belong to the different families. In order to perform thealignments a critical number of peptide sequences are required. Thus thefamilies investigated are the thrombospondin-1 containing peptides andthe peptides that belong to the C—X—C chemokines family. This analysiswas also extended to the families of collagen and TIMPs. In order torepresent the motifs single letter abbreviations of the amino acids thatare common and the letter “X” to denote a non-common amino acid thatintervenes the common letters. If there is more than one non-commonamino acid in between, the letter “X” is followed by the number of thenon-common amino acids. For example, if there are three non-common aminoacids between two conserved letters, this is denoted “a-X3-b”, where aand b are conserved residues. This notation is commonly used torepresent motifs.

Initially, multiple sequence alignments were performed with peptidesshowing experimentally demonstrated anti-angiogenic activity. Thiscalculation was subsequently extended to all the theoretically predictedfragments to determine whether the motifs calculated for theexperimentally tested fragments are conserved and reproduced in all ofthe anti-angiogenic predictions. The results of this analysis areorganized by protein family.

Thrombospondin-1 (TSP-1) Domain Containing Peptides

Of the thirty-one predicted and experimentally tested TSP-1 containingshort peptides, twenty-nine share a global 4 residue common motif whichis the X2-W—X2-C—X3-C—X2-G-X7 (SEQ ID NO: 180), or W—X2-C—X3-C—X2-G (SEQID NO: 161) after removing the uncommon edges, resulting in the genericTSP-1 containing 20-mer (FIG. 39). The first amino acid that succeedsthe first cysteine of the motif, or the seventh amino acid of thesequence can alternate between T, S and N. Thus a more genericdescription of this motif is X2-W—X2-C-(T/S/N)—X2-C—X2-G-X7 (SEQ ID NO:181) with threonine or serine the most abundant alteration for theseventh amino acid position.

By altering the threshold of the conserved amino acids that are commonamong the sequences of the predicted peptides subsets of peptidefamilies having individual common motifs of greater length than theglobal 4-letter motif are identified. The threshold is defined as thepercentage of the peptides that share a common motif. Such a subgroup ofpeptides is one that consists of 18 TSP-1 containing predictions(threshold 60%) that share a seven amino acid long common motif. Themotif is the X2-W—X2-C—S—X2-C-G-X1-G-X₃—R—X₃ (SEQ ID NO: 182). A commonalteration occurs in the 19^(th) amino acid which can be either anarginine or a valine with arginine the most abundant amino acid. In thatcase the motif is written X2-W—X2-C—S—X2-C-G-X1-G-X3-R—X1-(R/V)—X1 (SEQID NO: 183). Similarly the ninth amino acid can be altered by eitherarginine, serine or threonine. In that case the motif can be representedas X2-W—X2-C—S—X1-(S/R/T)-C-G-X1-G-X3-R—X1-(R/V)—X₁ (SEQ ID NO: 184)with threonine the most abundant amino acid (FIG. 40A). Similarlyanother motif with 45% threshold, common in 13 sequences, is the 5letter motif X1-P—W—X2-C—X3-C—X2-G-X7 (SEQ ID NO: 185). The commonalterations of this motif can be described as(S/G/Q)-P—W—X2-C-(T/S)—X2-C-(G/S)—X1-G-X3-(R/S)—X3 (SEQ ID NO: 186)(FIG. 40B).

In addition to calculating the motifs that are present within thesequences of the predicted fragments one can analyze all the possibleamino acids that are present within the 29 peptide sequences from whichthe motifs were calculated. This 20-mer with all the possiblesubstitutions is presented in the following table (Table 4) along withthe frequencies that each amino acid is present in the 29 sequences.

TABLE 4 The TSP-1 containing 20-mer with all the possible amino acidsubstitutions (SEQ ID NO: 162) AA1 AA2 AA3 AA4 AA5 AA6 AA7 AA8 AA9 AA10AA11 S (9) P (13) W (29) S (14) P (9) C (29) S (26) V (7) T (15) C (29)G (26) T (9) E (5) T (5) A (5) N (2) A (6) S (10) S (2) G (6) S (3) G(5) Q (4) T (1) R (5) R (3) N (1) Q (2) A (2) E (2) D (3) K (4) N (1) A(1) Q (1) D (1) E (3) G (2) K (1) R (1) K (1) S (2) A (1) R (1) T (2) V(1) E (1) AA12 AA13 AA14 AA15 AA16 AA17 AA18 AA19 AA20 G (10) G (29) V(8) Q (11) T (10) R (26) S (5) R (15) R (1) K (4) I (4) S (7) F (4) S(2) T (5) V (1) R (4) M (3) R (6) K (3) Q (1) V (5) M (4) T (3) K (2) Q(3) R (3) T (2) H (2) Y (2) S (3) H (3) L (2) A (1) A (1) L (2) E (2) D(1) E (1) E (1) Q (2) S (1) F (1) M (1) A (1) P (1) K (1) N (1) I (1) R(1) V (1) S (1) Q (1) W (1) Y (1)

The above motifs, for both the TSP-1 containing proteins were identifiedfrom the sequences of the peptide fragments that have already beenexperimentally tested in the proliferation assay. The specific approachfor identification of motifs within groups of sequences can begeneralized for the case of all the theoretically predictedanti-angiogenic fragments. For the TSP-1 containing fragments themultiple sequence alignment calculations are repeated, but now all ofthe theoretically predicted fragments are included.

For the cases of all the theoretically predicted TSP-1 containingproteins, multiple sequence alignment yielded a common motif within 97%of all the tested sequences. This motif, as described above,W—X2-C—X3-C—X2-G (SEQ ID NO: 161) (FIG. 41) and a generic 20-mer can beexpressed as X2-W—X2-C—X3-C—X2-G-X7 (SEQ ID NO: 180). It is interestingthat this motif is not a characteristic of only the TSP-1 domains, inother words it is not a signature domain of TSP-1 proteins. Rather, itappears to be present not only in a subset of TSP-1 proteins that areassociated with anti-angiogenic activity, but also within the type-2thrombospondin containing proteins (TSP-2) that are associated withanti-angiogenic activity. Thus, the W—X2-C—X3-C—X2-G (SEQ ID NO: 161)motif is associated with anti-angiogenic activity that spans at leasttwo protein families (i.e., TSP-1 and TSP-2 containing proteins).Moreover, in both experimentally tested fragments and in theoreticallypredicted fragments, the amino acid following the first cysteine of themotif can alternate between T, S and N. Thus a more specific descriptionof the motif is the W—X2-C-(T/S/N)—X2-C—X2-G (SEQ ID NO: 163) withserine and threonine being the predominant amino acids in the positionfollowing the first cysteine.

A common alteration occurs in the 19^(th) amino acid of the 20-mer whichcan be either an arginine or a valine with arginine the most abundantamino acid. In that case the motif is writtenX2-W—X2-C-(T/S/N)—X2-C—X2-G-X5-(R/V)—X (SEQ ID NO: 187).

Peptides Derived from C—X—C Chemokines

Based on the six predicted and experimentally tested C—X—C chemokines,all of them contain a six amino acid common motif. This motif can bedescribed as X-G-X3-C-L-X—P—X10-K—X-L (SEQ ID NO: 188) (FIG. 42). Thereare few common alterations that occur within the sequences of thepredicted fragments. For all those cases the motif can be re-written as(N/D)-G-(R/K)—X2—C-L-(N/D)-P—X2-(P/N)—X2-(K/Q)-(K/Q)-(I/V)—(I/V)-(E/Q)-K—X-L(SEQ ID NO: 173).

The generic 22-mer of the predicted C—X—C chemokines including all thepossible substitutions is presented in the following table (Table 5).

TABLE 5 The C-X-C chemokine 22-mer with all the possible amino acidsubstitutions (SEQ ID NO: 171) AA1 AA2 AA3 AA4 AA5 AA6 AA7 AA8 AA9 AA10AA11 AA12 AA13 N (4) G (6) R (3) K (3) A (2) C (6) L (6) D (4) P (6) A(2) A (3) P (6) F (2) D (2) K (3) E (2) I (2) N (2) E (2) S (2) I (1) Q(1) L (1) D (1) E (1) M (1) V (1) K (1) R (1) W (1) AA14 AA15 AA16 AA17AA18 AA19 AA20 AA21 AA22 V (3) K (4) K (5) I (3) I (4) E (3) K (6) I (3)L (6) L (2) Q (2) R (1) V (3) V (2) Q (3) F (1) I (1) K (1) M (1)

The calculations were repeated using all of the theoretically predictedC—X—C chemokines. These calculations also identified theX-G-X3-C-L-X—P—X10-K—X-L (SEQ ID NO: 188) motif as predicted by theexperimentally tested short fragments with minimal alterations (FIG.43).

All the theoretically predicted C—X—C chemokines include a generic22-mer that can be described as follows:

(SEQ ID NO: 172)(N/D/K)-G-X3-C-L-(D/N)-(P/L)-X5-(K/Q)-(K/R/N)-(I/V/L)-(I/V/L)-X6.This analysis indicates that the anti-angiogenic activity of the longerpredicted fragments is localized to the sites where the above-identifiedmotif resides.

Collagen Derived Peptides

The same procedure used to identify anti-angiogenic motifs in the C—X—Cchemokines can be used to identify anti-angiogenic motifs present incollagen related fragments. All the theoretically predicted fragmentswere used to identify two predominant anti-angiogenic motifs. The mostabundant and best characterized includes a conserved 4-amino acidrepeat: C—N—X3-V—C (SEQ ID NO: 174) (FIG. 44A). A set of two more motifsis shown in FIGS. 44B and 44C. The 4-letter motif appears upstream ofC—N—X3-V—C—X2-A-X—R—N-D-X—S—Y—W-L (SEQ ID NO: 176) (FIG. 44B). The4-residue motif appears downstream of the L-X2-F—S-T-X—P—F—X2-C—N—X3-V—C(SEQ ID NO: 177) (FIG. 44C) motif.

In addition to the aforementioned 7-mer there is another motif that ispresent in a smaller subset of collagen derived peptides. Those peptidesdo not include the C—N—X3-V—C (SEQ ID NO: 174). This motif is describedby the generic sequence X2-P—F—X-E-C—X-G-X5-A-N (SEQ ID NO: 189). Commonmodifications can be described by the sequence X2-P—F—(I/L)-E-C—X-GX—(R/G)-X—(Y/F)—(Y/F)-A-N (SEQ ID NO: 190) (FIG. 45).

Peptides Derived from TIMPs

Finally, motifs found within peptides derived from tissue inhibitors ofmetalloproteinases were identified. The peptides used included theloop-6 fragment of TIMP-2, which has been shown to have anti-angiogenicactivity. This analysis indicated that a motif having the sequenceE-C-L-W—X-D-X8-G-X—Y—X5-C (SEQ ID NO: 179) is present in TIMP peptidesas shown in Figure (FIG. 46).

No anti-angiogenic motifs have as yet been identified for the kringlecontaining peptides and the somatotropins.

Example 5 Results of Migration Experiments

An important constituent of the anti-angiogenic activity of an agent isits ability to reduce endothelial cell migration towards an attractantthat is present, such as a growth factor. Endothelial cell motility ormigration can be assessed using the modified Boyden chamber technique(Auerbach et al., Cancer Metastasis Rev, 19:167-72, 2000; Auerbach etal., Clin Chem, 49:32-40, 2003; Taraboletti and Giavazzi, Eur J Cancer,40:881-9, 2004).

In the current example, a modified Boyden chamber assay was used to testendothelial cell migration from one side of the chamber in the presenceof an activator. In brief, the lower compartment of the Boyden chamberwas separated from the upper (containing the endothelial cells) by alaminin-coated polycarbonate filter with pores small enough to allowonly the active passage of the cells (3 μm pore size). The cells wereapplied to the upper compartment of the chamber. The cell seedingdensity is 300,000 cells/ml. Typically, a cell population of25,000-30,000 cells was applied to each well. Activators include but arenot limited to growth factors, such as vascular endothelial growthfactor and fibroblast growth factor-2 or conditioned medium (e.g. fromtumor cells or NIH-3T3 fibroblasts). In the current example VEGF wasused as an activator and was applied to a growth factor and serum freemedium. The concentration of the activator applied to the lower chamberis 20 ng/ml. The activator was applied alone as a positive control. Thetested anti-angiogenic peptides were applied to the lower chamber at 30μg/ml concentration along with 20 ng/ml of activator in the serum freemedium. As negative control only the serum free and growth factor freemedium were applied. Typically within 4-20 hours a sufficient number ofcells migrated through the filter to allow measurements to be taken. Inthis example, the cells were allowed to migrate for 16 hours.

The number of migrating cells was quantified using a cell-permeablefluorescent dye. A fluorescence plate reader was used to quantify themigrating cells by measuring the amount of fluorescence and directlycorrelating it to cell number. A decrease in cell migration identifies apeptide that inhibits angiogenesis. In the current example the cellswere stained with Calcein dye, 90 minutes prior the termination of theexperiment. Calcein is internalized by endothelial cells and the cellsthat migrated towards the lower chamber were counted by measuring thefluorescence at 485 nm excitation and 510 nm emission using afluorescence plate reader (Victor 3V, Perkin Elmer). The wells werefluorescent impermeable thus the fluorescence emission only from thecells that migrated towards the lower chamber was measured. Theintensity of the signal was directly proportional to the number ofendothelial cells that were present in the lower chamber. The resultswere scaled so that 100% represents the mean of the positive control,where the cells are migrating in the presence of VEGF (FIG. 47). Most ofthe tested peptides significantly reduced the migration of theendothelial cells in the presence of 20 ng/ml VEGF. The peptides appliedwere C4α6 (YCNINEVCHYARRNDKSYWL) (SEQ ID NO: 93), C4α5(LRRFSTMPFMFCNINNVCNF) (SEQ ID NO: 89), TIMP3 (ECLWTDMLSNFGYPGYQSKHYACI)(SEQ ID NO: 155), C4α5 (FCNINNVCNFASRNDYSYWL) (SEQ ID NO: 90), C4α6(ATPFIECSGARGTCHYFAN) (SEQ ID NO: 94), C4α4 (AAPFLECQGRQGTCHFFAN) (SEQID NO: 87), C4α4 (LPVFSTLPFAYCNIHQVCHY) (SEQ ID NO: 85), C4α4(YCNIHQVCHYAQRNDRSYWL) (SEQ ID NO: 86), C4α5 (SAPFIECHGRGTCNYYANS) (SEQID NO: 91), TIMP4 (ECLWTDWLLERKLYGYQAQHYVCM) (SEQ ID NO: 156).

Analysis of Long Peptides

Apart from the predicted short peptide fragments that are produced usingthe aforementioned solid phase synthesis technique, there is apopulation of predicted anti-angiogenic fragments with lengths spanningfrom 55 to 134 amino acids. Based on the properties of knownanti-angiogenic peptides, short domains of approximately 25 amino acidshaving anti-angiogenic activity are expected to reside within theselonger sequences. In order to identify the domain, where theanti-angiogenic activity is localized, a “shotgun” methodology similarto the one used in proteomics approaches is used to localize theactivity within a shorter domain of the initially predicted longfragment. According to this shotgun approach each of the longersequences is partitioned into multiple sequential shorter fragments ofapproximately 25 amino acids length. In order to account for thepossibility that the active domain resides between these fragments, thesequential peptides are overlapping with a common sequence of length10-15 amino acids. Thus, one can effectively cover the whole sequence ofthe predicted long fragments with shorter peptides. However, thismethodology is time-consuming and costly.

A more systematic approach to identify and localize the active domainsof the longer fragments is achieved by combining the physico-chemicalcharacteristics of the peptides and the results of screening the shorterfragments. The cryptic fragments by definition are comprised ofsequences that are hidden in the three-dimensional structure of theprotein away from the rest of the protein environment. The parts of thesequence of a protein that are not exposed to an aqueous environment arehydrophobic. Presumably the cryptic fragments that are hidden fromaqueous environments are hydrophobic or partially hydrophobic. Thus, theactive domains of the cryptic fragments, which are evolutionary hiddenfrom the surrounding environment, are likely to be mostly hydrophobic.Based on this hypothesis, the locations of the active domains within thesequences of the larger predicted fragments are identified bycalculating the hydrophobicity of these large fragments. It is highlyprobable that the active domains are localized at locations where thesequence is hydrophobic. Using these methods, sequences havinganti-angiogenic activity are localized within the larger sequences ofpredicted fragments.

In addition to the information on the hydrophobicity, information fromsequences of the short peptides that have been tested to be active andthat contain or are parts of a specific conserved domain, in order toidentify the active domains of longer fragments that contain the sameconserved domain. By performing multiple sequence alignments to thesequences of the long predicted peptides with the sequences of theshorter fragments that are shown to be active, one can investigate theconservation of specific motifs that are common within the twopopulations of the peptides. If such motifs exist and are common betweenthose two populations then one can directly associate the location ofthe common motif in the longer fragments with the location of the activedomains. From there on one can investigate in the long fragments thesequences surrounding the common motif and identify the location of theactive domain.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1-11. (canceled)
 12. An isolated peptide or analog thereof comprising:(i) at least a fragment of a thrombospondin containing protein, whereinthe peptide reduces blood vessel formation in a tissue or organ andcomprises a sequence having at least 85% amino acid sequence identity toan amino acid sequence selected from the thrombospondin containingprotein listed in Table 1 (SEQ ID Nos: 1-76); (ii) a sequence having atleast 85% amino acid sequence identity to an amino acid sequenceselected from the group consisting of: THSD-1: QPWSQCSATCGDGVRERRR;(SEQ ID NO: 64) THSD-3: SPWSPCSGNCSTGKQQRTR; (SEQ ID NO: 65) THSD-6:WTRCSSSCGRGVSVRSR; (SEQ ID NO: 66) CILP: SPWSKCSAACGQTGVQTRTR;(SEQ ID NO: 39) WISP-1: SPWSPCSTSCGLGVSTRI; (SEQ ID NO: 74) WISP-2:TAWGPCSTTCGLGMATRV; (SEQ ID NO: 75) WISP-3: TKWTPCSRTCGMGISNRV;(SEQ ID NO: 76) F-spondin: SEWSDCSVTCGKGMRTRQR; (SEQ ID NO: 73)F-spondin: WDECSATCGMGMKKRHR; (SEQ ID NO: 72) CTGF: TEWSACSKTCGMGISTRV;(SEQ ID NO: 41) fibulin-6: ASWSACSVSCGGGARQRTR; (SEQ ID NO: 45)fibulin-6: QPWGTCSESCGKGTQTRAR; (SEQ ID NO: 44) fibulin-6:SAWRACSVTCGKGIQKRSR; (SEQ ID NO: 43) CYR61: TSWSQCSKTCGTGISTRV;(SEQ ID NO: 42) NOVH: TEWTACSKSCGMGFSTRV; (SEQ ID NO: 46) UNC5-C:TEWSVCNSRCGRGYQKRTR; (SEQ ID NO: 70) UNC5-D: TEWSACNVRCGRGWQKRSR;(SEQ ID NO: 71) SCO-spondin: GPWEDCSVSCGGGEQLRSR; (SEQ ID NO: 63)Properdin: GPWEPCSVTCSKGTRTRRR; (SEQ ID NO: 49) C6: TQWTSCSKTCNSGTQSRHR;(SEQ ID NO: 38) ADAMTS-like-4: SPWSQCSVRCGRGQRSRQVR; (SEQ ID NO: 69)ADAMTS-4: GPWGDCSRTCGGGVQFSSR; (SEQ ID NO: 7) ADAMTS-8:GPWGECSRTCGGGVQFSHR; (SEQ ID NO: 14) ADAMTS-16: SPWSQCTASCGGGVQTR;(SEQ ID NO: 24) ADAMTS-18: SKWSECSRTCGGGVKFQER; (SEQ ID NO: 29)semaphorin 5A: GPWERCTAQCGGGIQARRR; (SEQ ID NO: 60) semaphorin 5A:SPWTKCSATCGGGHYMRTR; (SEQ ID NO: 61) semaphoring 5B:TSWSPCSASCGGGHYQRTR; (SEQ ID NO: 62) papilin: GPWAPCSASCGGGSQSRS;(SEQ ID NO: 48) papilin: SQWSPCSRTCGGGVSFRER; (SEQ ID NO: 47) ADAM-9:KCHGHGVCNS; (SEQ ID NO: 157) and ADAM-12: MQCHGRGVCNNRKN, (SEQ ID NO: 2)

wherein A is alanine; I is isoleucine; M is methionine; H is histidine;Y is tyrosine; K is lysine; W is tryptophan; C is cysteine, T isthreonine, S is serine; N is asparagine; G is glycine; R is arginine; Vis valine, P is proline, and Q is glutamine wherein the peptide reducesblood vessel formation in a cell, tissue or organ; (iii) the amino acidsequence G-X₃—C-L-X—P—X₁₀—K—X-L (SEQ ID NO: 170), wherein X denotes avariable amino acid; G denotes Glycine; C denotes cysteine; L denotesleucine; P denotes proline; K is lysine; and the peptide reduces bloodvessel formation in a cell, tissue or organ; (iv) a 22 amino acidsequence having positions AA1-AA22 (SEQ ID NO: 171), wherein: AA1 is X,N or D; AA2 is G; AA3 is X, R or K; AA4 is X, K, E, or Q; AA5 is X, A,I, L, or V; AA6 is C; AA7 is L; AA8 is X, D or N; AA9 is P; AA10 is X,A, E, D, or K; AA11 is X, A, S, or E; AA12 is P; AA13 is X, F, I, M, R,or W; AA14 is X, V, L, or I; AA15 is X, K or Q; AA16 is X, K or R; AA17is X, I or V; AA18 is X, I or V; AA19 is X, E or Q; AA20 is K; AA21 isX, I, F, K, or M; and AA22 is L; wherein X denotes a variable aminoacid; A is alanine; I is isoleucine; F is phenylalanine; D is asparticacid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W istryptophan; C is cysteine, T is threonine, S is serine; N is asparagine;G is glycine; R is arginine; V is valine, P is proline, and Q isglutamine; and wherein the peptide reduces blood vessel formation in acell, tissue or organ; (v) an amino acid sequence selected from thegroup consisting of: (SEQ ID NO: 170) G-X₃-C-L-X-P-X₁₀-K-X-L;(SEQ ID NO: 172)(N/D/K)-G-X₃-C-L-(D/N)-(P/L)-X₅-(K/Q)-(K/R/N)-(I/V/L)-(I/V/L)-X₆; and(SEQ ID NO: 173)(N/D)-G-(R/K)-X₂-C-L-(N/D)-P-X₂-(P/N)-X₂-(K/Q)-(K/Q)-(I/V)-(I/V)-(E/Q)-K-X-L;

(vi) a sequence that has at least 85% amino acid sequence identity to aCXC Chemokine protein listed in Table 1 (SEQ ID Nos: 95-116); (vii) asequence having at 85% identity to an amino acid sequence selected fromthe group consisting of: ENA-78: NGKEICLDPEAPFLKKVIQKILD;(SEQ ID NO: 95) CXCL6: NGKQVCLDPEAPFLKKVIQKILDS; (SEQ ID NO: 98) CXCL1:NGRKACLNPASPIVKKIIEKMLNS; (SEQ ID NO: 102) Gro-γ:NGKKACLNPASPMVQKIIEKIL; (SEQ ID NO: 106) Beta thromboglobulin/CXCL7:DGRKICLDPDAPRIKKIVQKKL, (SEQ ID NO: 114) Interleukin 8 (IL-8)/CXCL8:DGRELCLDPKENWVQRVVEKFLK, (SEQ ID NO: 110) GCP-2:NGKQVCLDPEAPFLKKVIQKILDS, (SEQ ID NO: 98)

wherein A is alanine; I is isoleucine; F is phenylalanine; D is asparticacid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W istryptophan; C is cysteine, T is threonine, S is serine; N is asparagine;G is glycine; R is arginine; V is valine, P is proline, and Q isglutamine; and wherein the peptide reduces blood vessel formation in acell, tissue or organ; (viii) a peptide comprises at least a fragment ofa C—X—C chemokine protein of Table 1; (ix) an amino acid sequenceC—N—X₃—V—C (SEQ ID NO: 174) or P—F—X-E-C—X-G-X₅-A-N (SEQ ID NO: 175),wherein X denotes a variable amino acid; F is phenylalanine; C iscysteine, N is asparagine; G is glycine; V is valine, P is proline, andQ is glutamine wherein the peptide reduces blood vessel formation in acell, tissue or organ; (x) a sequence comprising at least a fragment ofa type IV collagen C4 polypeptide, wherein the peptide reduces bloodvessel formation in a cell, tissue or organ and comprises a sequencehaving at least 85% identity to an amino acid sequence selected fromcollagen proteins listed in Table 1; (xi) one of the following aminoacid sequences: C—N—X₃—V—C—X2-A-X—R—N-D—X—S—Y—W-L (SEQ ID NO: 176);L-X2-F—S-T—X—P—F—X₂—C—N—X₃—V—C (SEQ ID NO: 177), wherein the peptidereduces blood vessel formation in a cell, tissue or organ; (xii) anamino acid sequence P—F—(I/L)-E-C—X-G-X—(R/G)-X—(Y/F)—(Y/F)-A-N (SEQ IDNO: 178), wherein the peptide reduces blood vessel formation in a cell,tissue or organ; (xiii) a sequence having at least 85% amino acidsequence identity to an amino acid sequence selected from the groupconsisting of Alpha 6 fibril of type 4 collagen: YCNINEVCHYARRNDKSYWL;(SEQ ID NO: 93) Alpha 5 fibril of type 4 collagen: LRRFSTMPFMFCNINNVCNF;(SEQ ID NO: 89) Alpha 4 fibril of type 4 collagen: AAPFLECQGRQGTCHFFAN;(SEQ ID NO: 87) Alpha 4 fibril of type 4 collagen: LPVFSTLPFAYCNIHQVCHY;(SEQ ID NO: 85) and Alpha 4 fibril of type 4 collagen:YCNIHQVCHYAQRNDRSYWL, (SEQ ID NO: 86)

wherein A is alanine; I is isoleucine; F is phenylalanine; D is asparticacid; M is methionine; H is histidine; Y is tyrosine; K is lysine; W istryptophan; C is cysteine, T is threonine, S is serine; N is asparagine;G is glycine; R is arginine; V is valine, P is proline, and Q isglutamine wherein the peptide reduces blood vessel formation in a cell,tissue or organ; (xiv) an amino acid sequence E-C-L-W—X-D-X₈-G-X—Y—X₅—C(SEQ ID NO: 179), wherein the peptide reduces blood vessel formation ina cell, tissue or organ; (xv) a peptide comprising or consistingessentially of twenty-three amino acids of a TIMP protein listed inTable 1; (xvi) a peptide comprising or consisting essentially of atleast twenty-four amino acids of a TIMP protein listed in Table 1;(xvii) a peptide comprising or consisting essentially of 30 amino acidsof a TIMP protein listed in Table 1; (xviii) a peptide comprising orconsisting essentially of an amino acid sequence having at least 85%amino acid sequence identity to ECLWTDMLSNFGYPGYQSKHYACI (SEQ ID NO:155), wherein the peptide reduces blood vessel formation in a cell,tissue or organ; (xix) a fragment of a TIMP, wherein the peptidecomprises a sequence having at least 85% amino acid sequence identity toan amino acid sequence selected from TIMP protein listed in Table 1, andwherein the peptide reduces blood vessel formation in a cell, tissue ororgan; (xx) a fragment of an amino acid sequence selected from the groupconsisting of SEQ ID Nos. 1-156; (xxi) a peptide comprising orconsisting essentially of an amino acid sequence having at least 85%identity to an amino acid sequence selected from the group consisting ofSEQ ID Nos. 1-156 (xxii) a peptide or analog thereof comprising theamino acid sequence W—X₂—C—X₃—C—X₂-G (SEQ ID NO: 161), wherein X denotesa variable amino acid; W is tryptophan; C is cysteine, G is glycine; andwherein the peptide reduces blood vessel formation in a cell, tissue ororgan; (xxiii) a peptide comprising or consisting essentially of 10amino acids of a thrombospondin containing protein of Table 1 (SEQ IDNos: 1-76); or (xxiv) a peptide or analog thereof comprising a 20 aminoacid (AA) sequence having positions AA1-AA20 (SEQ ID NO: 162), wherein:AA1 is X, S, T, G, Q, or A; AA2 is X, P, E, S, A, Q, or K; AA3 is W; AA4is X, S, T, G, E, D, R, or A; AA5 is X, P, A, Q, D, E, K, R, or V; AA6is C; AA7 is X, S, N, or T; AA8 is X, V, A, R, K, G, S, T, or E; AA9 isX, T, S, R, or N; AA10 is C; AA11 is X, G, S, or N; AA12 is X, G, K, R,M, T, L, D, S, or P; AA13 is G; AA14 is X, V, I, M, T, H, A, E, F, K, R,S, Q, W, or Y; AA15 is X, Q, S, R, K, Y, or A; AA16 is X, T, F, K, Q, S,L, E, M, N, or V; AA17 is X, R, S, or Q; AA18 is X, S, T, V, R, H, E, Q,A, or I; AA19 is X, R, or V; and AA20 is R; wherein X denotes a variableamino acid; W is tryptophan; C is cysteine, T is threonine, S is serine;N is asparagine; G is glycine; R is arginine; V is valine, P is proline,and Q is glutamine; and wherein the peptide reduces blood vesselformation in a cell, tissue or organ. 13-38. (canceled)
 39. The isolatedpeptide of claim 12, wherein the peptide comprises at least onemodification. 40-42. (canceled)
 43. A peptide conjugate comprising thepeptide of claim 12 conjugated to an agent that specifically binds atumor marker or endothelial cell marker. 44-46. (canceled)
 47. Anisolated nucleic acid molecule encoding the peptide of claim
 12. 48. Anexpression vector comprising the nucleic acid molecule of claim 47,wherein the nucleic acid molecule is positioned for expression. 49.(canceled)
 50. A host cell comprising the peptide of claim 12 or anucleic acid molecule encoding the peptide. 51-53. (canceled)
 54. Amethod of reducing blood vessel formation in a tissue or organ, themethod comprising contacting an endothelial cell, or a tissue or organcomprising an endothelial cell with an effective amount of the peptideof claim 12 or a conjugate thereof, thereby reducing blood vesselformation in the tissue or organ.
 55. A method of reducing endothelialcell proliferation, migration, survival, or stability in a tissue ororgan, the method comprising contacting tissue or organ comprising anendothelial cell with an effective amount of the peptide of claim 12 ora conjugate thereof, thereby reducing endothelial cell proliferation,migration, survival, or stability in the tissue or organ.
 56. A methodof increasing endothelial cell death in a tissue or organ, the methodcomprising contacting a tissue or organ comprising an endothelial cellwith an effective amount of the peptide of claim 12 or a conjugatethereof, thereby increasing endothelial cell death in the tissue ororgan.
 57. (canceled)
 58. A method of reducing blood vessel formation ina tissue or organ the method comprising: (a) contacting the tissue, ororgan with a vector encoding the polypeptide of claim 12 or a conjugatethereof; and (b) expressing the polypeptide in a cell of the tissue ororgan, thereby reducing blood vessel formation in the tissue or organ.59-63. (canceled)
 64. A method for decreasing blood vessel formation ina subject in need thereof, the method comprising administering aneffective amount of the peptide of claim 12 or a conjugate thereof tothe subject, thereby decreasing blood vessel formation.
 65. A method ofreducing endothelial cell proliferation, migration, survival, orstability in a subject in need thereof, the method comprisingadministering an effective amount of the peptide of claim 12 or aconjugate thereof to the subject, thereby reducing endothelial cellproliferation, migration, survival, or stability in the tissue or organ.66. A method of increasing endothelial cell death in a subject in needthereof, the method comprising administering an effective amount of apeptide of claim 12 or a conjugate thereof to the subject, therebyincreasing endothelial cell death in the subject. 67-81. (canceled) 82.A pharmaceutical composition comprising an effective amount of anisolated peptide of claim 12 or a conjugate thereof in apharmacologically acceptable excipient.
 83. A pharmaceutical compositioncomprising an effective amount of a nucleic acid molecule or portionthereof encoding a peptide of claim 12 or a conjugate thereof in apharmacologically acceptable excipient.
 84. A pharmaceutical compositioncomprising i) an isolated polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID Nos. 78-80, 82, 83, 85-87,89-91, 93, and 94, and ii) one or more peptides of SEQ ID Nos. 1-156.85. A method for identifying an amino acid sequence of interest, themethod comprising: (a) identifying an initial polypeptide or fragmentthereof having amino acid sequence identity to a reference sequencehaving a biological function of interest; (b) generating a randomsequence comprising the amino acid composition of the initialpolypeptide of interest and comparing the random sequence to thereference sequence to determine amino acid sequence identity, whereinsaid amino acid sequence identity determines a random sequence cut-offvalue; (c) comparing the sequence identity of step a to the randomsequence cut-off value of set b, wherein a sequence identity that issignificantly greater than the random sequence cut-off value identifiesan amino acid sequence of interest. 86-91. (canceled)